Inhibitors of valosin-containing protein and methods of use

ABSTRACT

A method of inhibiting aberrant valosin-containing protein (VCP) accumulation in the mitochondria of a nerve cell includes administering to the nerve cell a therapeutic agent that inhibits the binding or complexing of VCP with a polyglutamine protein.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.62/113,063, filed Feb. 6, 2015, the subject matter of which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

This application relates to compositions and methods for inhibitingaberrant Valosin-Containing Protein (VCP) accumulation in themitochondria of nerve cells, and particularly relates to compositionsand methods for treating diseases or disorders associated with aberrantVCP accumulation in the mitochondria of nerve cells.

BACKGROUND

There are a number of neurodegenerative polyglutamine diseases, forexample Huntington's disease, spinocerebellar ataxia, and spinobulbarmuscular atrophy (Kennedy's Disease), which are characterized byexpanded genomic CAG sequences resulting in the synthesis andaccumulation of polyglutamine tracts in brain proteins of unknownfunction (e.g., Huntingtin in Huntington's disease and ataxin inspinocerebellar ataxias) that are responsible for the neurologicproblem. The CAG codon is translated into glutamine (Q). Proteins withexpanded polyglutamine domains aggregate and aggregation is a pathologichallmark of the polyglutamine repeat diseases (Hackam, A. S. et al. JCell Biol 141, 1097-1105 (1998); Perez, M. K. et al. J Cell Biol 143,1457-1470 (1998)). These polyglutamine length-dependent properties mayarise from the ability of long polyglutamine domains to adopt uniquethree-dimensional conformations and serve to confer the disease proteinswith a pathologic gain-of-function (Perutz, M. F. Trends Biochem Sci 24,58-63 (1999); Lansbury, P. T. J. Proc Natl Acad Sci USA 96, 3342-3344(1999)).

All diseases in the CAG repeat family show genetic anticipation, meaningthe disease usually appears at an earlier age and increases in severitywith each generation. Genetic anticipation is linked to increasingnumbers of CAG repeats, which result from expansion of the unstable CAGsequence when reproductive cells divide to form eggs and sperm. Ingeneral, neurodegenerative disorders are progressive (i.e., theirsymptoms are not apparent until months or more commonly years after thedisease has begun), and caused by an initial reduction of neuronalfunction, followed by a complete loss of function upon neuronal death.

Huntington's Disease (HD) is a devastating, degenerative brain disorderfor which there is, at present, no effective treatment or cure. HDslowly diminishes the affected individual's ability to walk, think, talkand reason. Eventually, the person with HD becomes totally dependentupon others for his or her care. Huntington's Disease profoundly affectsthe lives of entire families: emotionally, socially and economically.Early symptoms of Huntington's Disease may affect cognitive ability ormobility and include depression, mood swings, forgetfulness, clumsiness,involuntary twitching and lack of coordination. As the diseaseprogresses, concentration and short-term memory diminish and involuntarymovements of the head, trunk and limbs increase. Walking, speaking andswallowing abilities deteriorate. Eventually the person is unable tocare for him or herself. Death follows from complications, such aschoking, infection or heart failure. HD typically begins in mid-life,between the ages of 30 and 45, though onset may occur as early as theage of 2. Children who develop the juvenile form of the disease rarelylive to adulthood. HD affects males and females equally and crosses allethnic and racial boundaries. Each child of a person with HD has a 50/50chance of inheriting the fatal gene. HD is an autosomal dominantcondition and thus everyone who carries the gene will develop thedisease.

The Huntington's Disease (HD) gene was mapped to chromosome 4p16.3 in1983 but eluded identification until 1993. When finally identified, thegene (IT15) was found to contain a CAG repeat within its 5′-end codingsequence (Cell 72:971-983). This CAG repeat is expanded in individualswith HD who may or may not be symptomatic. However, the presence of aCAG repeat expansion is found in virtually all symptomatic HDindividuals (N. Engl. J. Med. 330:1401-1406).

Normal HD gene CAG repeats range from 10-29 repeats. Some normalindividuals (<1%) have been found with intermediate HD gene CAG repeatsof 30-35 repeats. Individuals affected with HD typically have at leastone HD gene CAG repeat of 36 repeats or greater. It was also found thatin a few rare instances (10 cases) individuals having repeats of 36-39repeats had remained asymptomatic by standard clinical criteria atadvanced age. In one exceptional case, a 95 year old patient had 39repeats (Rubinsztein et. al., 1996; Am. J. Hum. Genet. 59:16-22). Thereis a tendency to an earlier age-of-onset of HD symptoms with increasingCAG repeat number. A review of 1,049 people (the majority of whom weresymptomatic) has provided a determination of the likelihood of anage-of-onset for a given CAG repeat size for repeats between 39-50repeats (Brinkman et al., 1997; Am. J. Hum. Genet. 60:1202-1210). Thepolyglutamine expansion results in the formation of insoluble, highmolecular weight protein aggregates similar to those seen in Alzheimer'sdisease (Scherzinger et al., Cell 90:549-558 [1997]). Postmortemexamination of the brains of patients suffering from Huntington'sdisease revealed that CAG repeat length positively correlates with thedegree of DNA fragmentation within the afflicted striatum (Butterworthet al., Neurosci., 87:49-53 [1998]), indicating that neuronaldegeneration observed in Huntington's disease may also occur through anapoptotic process.

Currently, physicians may prescribe a number of medications to helpcontrol emotional and movement problems associated with polyglutaminedisorders caused by expanded genomic CAG nucleotides. Such medicationsinclude antipsychotic drugs, such as haloperidol, or other drugs, suchas clonazepam, to alleviate choreic movements and also to help controlhallucinations, delusions, and violent outbursts; fluoxetine,sertraline, nortriptyline, or other compounds may be prescribed fordepression. Tranquilizers can help control anxiety and lithium may beprescribed to combat pathological excitement and severe mood swings. Itis important to remember however, that while medicines may help keepthese clinical symptoms under control, there is currently no treatmentto stop or reverse the course of the disease.

Remacemide and Coenzyme Q10 have been tested for the treatment of HD buta large-scale clinical trial that tested the ability of theseinvestigational drugs to slow the progression of Huntington's diseaseshowed that neither drug resulted in any significant improvement for thepatients. Remacemide blocks a neurotransmitter in the brain (the NMDAglutamate receptor) which has long been suspected of contributing to thedeath of brain cells in Huntington's disease. Coenzyme Q10 is asubstance that occurs naturally in the body and plays a role in thefunction of mitochondria, the energy factories of human cells. It isalso an anti-oxidant, meaning that it can neutralize potentiallyinjurious oxygen-containing chemicals called free radicals, which mayplay a role in the nerve cell death that occurs in Huntington's disease.After one year of treatment, the disease seemed to progress more slowlyin patients treated with Coenzyme Q10, however, the investigatorsconcluded that overall the results were inconclusive as to whether thereis real benefit from this drug (Neurology, Aug. 14, 2001; 57: 397).

SUMMARY

Embodiments described herein relate to methods of inhibitingvalosin-containing protein (VCP) accumulation in mitochondria of a nervecell and particularly relates to methods of treating a disorderassociated with aberrant VCP accumulation by polyglutamine proteins inmitochondria of nerves cells in a subject in need thereof.

The methods can include administering to nerve cells of the subject atherapeutic agent that inhibits the binding or complexing of VCP withpolyglutamine proteins. In some embodiments, the disorder can include aneurodegenerative disorder, such as a polyglutamine neurodegenerativedisease. In other embodiments, the polyglutamine protein is mutanthuntingtin protein (mtHtt) and the disorder is Huntington's disease.

In other embodiments, the therapeutic agent can include a therapeuticpeptide. The therapeutic peptide can have at least about 75% sequenceidentity to about 8 to about 10 consecutive amino acids of aninteraction site of VCP with the polyglutamine protein. In someembodiments, the therapeutic peptide can have an amino acid sequencethat is at least about 75% identical to SEQ ID NO: 4. For example, thetherapeutic peptide can have an amino acid sequence of SEQ ID NO: 3.

In other embodiments, the therapeutic agent can include a transportmoiety that is linked to the therapeutic peptide and facilitates uptakeof the therapeutic peptide by the nerve cell. The transport moiety canbe, for example, an HIV Tat transport moiety.

In other embodiments, the therapeutic agent can be administeredsystemically to the subject being treated. For example, a therapeuticagent that includes a therapeutic peptide having an amino acid sequenceof SEQ ID NO: 3, which is linked to a HIV Tat transport moiety, can beadministered intravenously to a subject with Huntington's disease.

The amount of the therapeutic agent that is administered to the subjectcan be an amount effective to increase plasma levels of NAD+, FAD,and/or citrate in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A-H) illustrate: VCP was recruited to mitochondria by mtHtt andbound to mitochondria-associated mtHtt in vitro and in vivo. (A)Affinity purification followed by tandem mass spectrometry analysis wasconducted to identify mtHtt-binding proteins on mitochondria in HDknock-in mouse HdhQ7 and HdhQ111 striatal cells. Shown is the molecularand cellular function of the mtHtt mitochondrial interactome in HdhQ111cells. Among these proteins, VCP was the leading candidate for anmtHtt-binding protein. (B) Mitochondrial and ER fractions were isolatedfrom mouse HdhQ7 and HdhQ111 striatal cells. Protein levels of VCP wereanalyzed by Western blotting. VDAC and WFS1 were used as loadingcontrols of mitochondria and ER. Data are mean±SE of three independentexperiments. (C) Control siRNA (Con) and Htt siRNA (siHTT) weretransfected in HdhQ7 and HdhQ111 cells, respectively. VCP levels weredetermined in mitochondrial fractions by Western blot analysis. VDAC wasa loading control. Data are mean±SE of three independent experiments.(D) HdhQ7 and HdhQ111 mouse striatal cells were stained with anti-Tom20(green, a mitochondrial marker) and anti-VCP (red) antibodies. Scalebars: 10 μm. VCP/Tom20 co-localization was examined using confocalmicroscopy. Pearson's co-efficiency was calculated. At least 100 cellsper group were counted by an observer blind to experimental conditions.Data are mean±SE of three independent experiments. (E) Mitochondria wereisolated from the striatum of either HD transgenic mice R6/2 at the ageof 9 weeks or YAC128 at the age of 6 months. n=6 mice/group. VCP levelswere determined by Western blot (loading control: VDAC). Data aremean±SE. (F) Paraffin-embedded sections (5 μm thick) of caudate nucleusfrom three HD patients (ID: 2982, 2983 and 5413) and three normalsubjects (ID: 623, 624 and 1533) were immune-stained with anti-VCP (red)and anti-Tom20 (green) antibodies. Localization of VCP on mitochondriawas examined using confocal microscopy. Pearson's co-efficiency wascalculated. Patient 2982 (56 years old, female), 2983 (30 years old,male), 5413 (43 years old, male) exhibited moderate neuronal loss incaudate nucleus and died of HD. The three normal subjects had no historyof HD and other neurological diseases. Subject 623 (85 years old, male)and 624 (62 years old, female) were died of otosclerosis and subject1533 (79 years old, female) died of spasmodic torticollis. The centralnervous system of the three normal subjects showed no pathognomonicchanges. Normal caudate nucleus and basal ganglia of these threesubjects were observed. (G) Mitochondria, ER and cytosolic fractions(Ct) of HdhQ7 and HdhQ111 mouse striatal cells were subjected toimmunoprecipitation (IP) with anti-VCP antibody, and immunoprecipitateswere analyzed by immunoblotting (IB) with anti-VCP and anti-MAB2166antibody (recognizes both wt and mtHtt, left panel) or anti-1C2 antibody(recognizes mtHtt, right panel). Note that polyQ protein above 250 kDawas shown in the right panel. Shown are representative blots from threeindependent experiments. (H) Mitochondria, ER, and cytosolic fractionswere isolated from striata of YAC128 and wildtype mice at the age of 6months. IP with anti-VCP antibody followed by anti-1C2 antibody oranti-EM48 antibody was performed. The right panel indicates the purityof ER and mitochondrial fractions isolated from YAC128 mouse striatum.WFS1 and VDAC were used to label ER and mitochondria, respectively. n=4mice/group.

FIGS. 2(A-F) illustrate the development of a peptide blocker of Htt/VCPbinding. (A) Sequence of homology between VCP (human, AAI21795) and Htt(human, NP_002102). Amino acids (SEQ ID NOs: 1-4) are represented by theone-letter code; stars (*) indicate identical amino acids; Columns (:)indicate high similarity between amino acids. Peptides HV1-4 correspondto these homologous regions. (B) Stick drawings of VCP and Htt maindomains. Highlighted in the same colors are the two regions of homologybetween the two proteins, regions HV-1 and HV-3 in Htt and thecorresponding regions HV-2 and HV-4 in VCP. (C) Mouse wild-type striatalcells were transfected with Myc control vector, Myc-full-length Htt with23 Q or 73Q (Myc-23Q FL or Myc-73Q FL) and GFP-VCP for 48 hoursfollowing the treatment with peptide HV-3 or control peptide TAT (3μM/day, each). The total lysates of cells were subjected to IP withanti-Myc antibodies followed by IB analysis with anti-VCP antibody.Shown are representative blots of three independent experiments. (D) HDmouse striatal cells were treated with the control peptide TAT or HV-3(3 μM/day for 3 days). The shown blots are from three independentexperiments. (E) YAC128 or wild-type mice were treated with controlpeptide TAT or peptide HV-3 (3 mg/kg/day) from the age of 3 months to 6months. n=6 mice/group. (F) HD R6/2 mice or wild-type mice were treatedwith control peptide TAT or peptide HV-3 (3 mg/kg/day) from the age of 5weeks to 9 weeks. n=6 mice/group. Mitochondrial fractions were isolatedfrom cells or striata of mice. VCP mitochondrial levels were determinedby western blot analysis. VDAC was used as a loading control. Data aremean±SE.

FIGS. 3(A-H) illustrate the peptide HV-3 treatment reduced mitochondrialdamage and cell death in HD cell cultures. Mouse HdhQ7 and HdhQ111striatal cells were treated with control peptide TAT or peptide HV-3 (3μM/day for 3 days). (A) Mitochondrial membrane potential was determinedby TMRM fluorescent dye. (B) Mitochondrial morphology was determined bystaining cells with anti-Tom20 antibody. The percentage of cells withfragmented mitochondria relative to total number of cells wasquantitated. For quantitation of imaging in above cells, at least 100cells per group were counted by an observer blind to experimentalconditions. (C) HD striatal cells were subjected to serum starvation for24 hours. High-mobility group protein B1 (HMGB1) release into culturemedium was determined by Western blot analysis with anti-HMGB1 antibody.(D) HD striatal cells were subjected to serum starvation for 24 hours.Cell death was determined by the release of lactate dehydrogenase (LDH).Control and HD patient-iPS cell derived neurons were treated withpeptide HV-3 or control peptide TAT at 1 μM/day for 5 days starting 30days after initiation of neuronal differentiation. (E) Left: Neuronswere stained with anti-DARPP-32 and anti-Tuj-1 antibodies to indicatemedium spiny neurons. Upper: a cluster of neurons; lower: individualneurons. (F) Quantitation of neurite length of medium spiny neurons. Atleast 50 neurons per group were counted by an observer blind toexperimental conditions. (G) Left: Mitochondrial membrane potential wasdetermined by TMRM fluorescent dye. Right: Mitochondria were stained byanti-Tom20 antibody. Mitochondrial length along neurites ofDARPP-32-positive neurons was quantitated. (H) Neuronal cell deathinduced by the withdrawal of the growth factor BDNF for 24 hours wasdetermined by the release of LDH. All the Data are mean±SE from at leastthree independent studies. Scale bars: 10 μm.

FIGS. 4(A-G) illustrate the treatment of HV-3 reduced excessivemitophagy in HD cell cultures and HD YAC128 mouse brains. (A) HdhQ7 andHdhQ111 cells were treated with control siRNA (con) or VCP siRNA (siVCP)for 48 hours. Mitochondria were isolated and LC3 mitochondrial levelswere determined by Western blot. The quantitation of LC3II levels onmitochondria is provided on the right. VADC was used here as a loadingcontrol. (B) Flag-VCP and GFP-LC3B were co-transfected into HeLa cells.Mitochondria were isolated after 36 hours of transfection. The GFP-LC3Blevels on mitochondria were examined by western blot analysis. VDAC wasused as a loading control. Histogram: quantitation of GFP-LC3Bmitochondrial protein level. HdhQ7 and HdhQ111 cells were treated withcontrol peptide TAT or peptide HV-3 (3 μM/day for 3 days). (C) HdhQ111cells were transfected with GFP-LC3B for 24 hours. The number ofGFP-LC3B puncta was quantitated and shown in the histogram. Scale bars:10 μm. (D) Enzyme activity of lysosomal cathepsin B was measured using acathepsin B assay kit. Control and HD patient-iPS cell derived neuronswere treated with peptide HV-3 or control peptide TAT at 1 μM/day for 5days starting 30 days after neuronal differentiation. (E) Mitochondrialmass was measured by the fluorescent density of Mitotracker green. (F)Lysosomal activity was examined by staining neurons with Lyso-ID Reddye. Scale bars: 10 μm. (G) YAC128 mice and wildtype mice were treatedwith control peptide TAT or peptide HV-3 (3 mg/kg/day) from the age of 3months to 9 months. Transmission electron microscope images of striatafrom 9-month-old wild-type mice and YAC128 mice was performed. Arrowsindicate mitophagosomes. Histogram: the number of mitophagosomes per 100μm² was counted and quantitated. Fifteen random areas in the striatum ineach animal were analyzed. All the data are mean±SE of three independentexperiments.

FIGS. 5(A-F) illustrate: VCP caused excessive mitophagy by binding toLC3. (A) Putative LIR sequences in VCP were aligned manually forcomparison with the classical LIR motifs of ATG32, FUNDC1 and p62. Theamino acids in blue indicate the conserved core residues of LIR. (B)GFP-LC3B was co-expressed with the indicated plasmids in HeLa cells.Mitochondrial lysates were subjected to immunoprecipitation (IP) withanti-GFP antibody, and immunoprecipitates were analyzed byimmunoblotting (IB) with anti-Myc and anti-GFP antibodies.Representative blots are from 3 independent experiments. (C) GFP-LC3Bwas co-transfected with the indicated plasmids in HeLa cells.Mitochondria were isolated and GFP-LC3B mitochondrial protein levelswere determined by Western blot. Data are mean±SE from 4 independentstudies. (D) HeLa cells were transfected with the indicated plasmids.Mitochondria were stained with an anti-Tom20 antibody. Mitochondrialmass was determined by quantitating fluorescent density of Tom20immunostaining. At least 100 cells per group were counted by an observerblind to experimental conditions. Data are mean±SE from 3 independentstudies. Primary rat striatal neurons (DIV 7) were transfected withflag-VCPmt plasmids for 3 days. (E) Neurons were stained with anti-Tom20(green) and anti-flag (red) antibodies. Mitochondrial morphology wasexamined by microscopy. (F) Medium spiny neurons were labeled withanti-DARPP-32 (green). Neuronal morphology was imaged and the neuritelength of medium spiny neurons was quantitated. At least 50 neurons pergroup were counted by an observer blind to experimental conditions.Scale bars: 10 μm. All the data are mean±SE from 3 independentexperiments.

FIGS. 6(A-E) illustrate: HV-3 treatment reduced motor deficits in bothR6/2 and YAC128 HD mice. HD R6/2 mice and wild-type littermates weretreated with either the control peptide or peptide HV-3 (at 3 mg/kg/day,subcutaneous administration with an Alzet osmotic pump) from 5 to 13weeks of age. (A) One hour of overall movement activity in R6/2 mice andwildtype littermates (total traveled distance, horizontal and verticalactivities) was determined by locomotion activity chamber at the age of13 weeks (n=15 mice/group). Hindlimb clasping was assessed with the tailsuspension test once a week from the ages of 8 to 11 weeks (n=15mice/group). *, p<0.05. Body weight (B) and survival (C) were recordedfrom the age of 5 weeks to 13 weeks (n=15 mice/group). *, p<0.05 vs. HDmice treated with control peptide TAT; repeated-measures two-way ANOVA.YAC128 mice and wild-type littermates were treated with the controlpeptide TAT or HV-3 peptides from the age of 3 months to the age of 12months. Mouse behavioral and HD-associated pathology were determinedevery three months after beginning treatment. (D) 24 hours of generalmotility of YAC128 mice and wildtype littermates was monitored by alocomotion activity chamber at the indicated age (n=15-20 mice/group).#, p<0.05 vs. wild-type mice treated with control peptide TAT; *, p<0.05vs. HD mice treated with control peptide TAT. (E) Rotarod performance ofYAC128 and wildtype mice was evaluated at the indicated age (n=15-20mice/group). #, p<0.05 vs. wild-type mice treated with control peptideTAT; *, p<0.05 vs. HD mice treated with control peptide TAT. All dataare expressed as mean±SE.

FIGS. 7(A-E) illustrate: HV-3 treatment reduced mitochondrial defectsand neuropathology in HD mice. (A) DARPP-32 protein levels weredetermined by Western blot of R6/2 (left) and YAC128 (right) mousestriatal extracts. Upper: representative immunoblotting (IB); Lower:histogram of quantification of DARPP-32 levels. Actin was used as aloading control. Data are mean±SE of 6 mice/group. (B) Photomicrographsof DARPP-32 immunostaining were obtained from the dorsolateral striatumof TAT- or HV-3-treated R6/2 mice. (C) Quantitation of DARPP-32immunodensity. Data are mean±SE of 6 mice. (D) Quantitation ofNeuN-immunopositive cells in the dorsolateral striatum. Data are mean±SEof 6 mice. (E) A summary scheme. VCP is selectively recruited to themitochondria by interacting with mitochondria-bound mtHtt.Mitochondria-accumulated VCP acts as a mitophagic adaptor to bind to theautophagosome component LC3 via an LC3 interacting region (LIR motif).As a result, mtHtt-induced VCP association with mitochondria causesexcessive mitophagy which results in mitochondrial mass loss,mitochondrial dysfunction and neuronal cell death. Blocking mtHtt to VCPbinding on mitochondria by a selective peptide HV-3 inhibits VCPmitochondrial accumulation, which reduces excessive mitophagy andsubsequent neuronal degeneration. Consequently, treatment with HV-3 bothin HD cultures and in HD animals reduces HD-associated neuropathology.

FIGS. 8(A-E) illustrate: GST or GST-VCP was incubated with total lysatesof mouse brain and the indicated peptides (3 μM) for 16 hours, followedby immunoblotting with anti-Htt antibodies. Representative blots arefrom three independent experiments. Quantitation of VCP/Htt binding wasshown below the blot as Mean of three independent experiments. *, p<0.05vs. TAT-treated group. (B) HEK293 cells were co-expressed GFP-VCP andMyc-73Q FL plasmids as indicated. After 48 hours incubation with theindicated peptides (3 μM/day, each), immunoprecipitation analysis wasperformed. The shown blots are from three independent experiments. (C)Upper: sequence of the HV-3 peptide and control peptide TAT. Lower: HV-3peptide sequence is highly conserved among species. (D) The HV-3 peptidewas docked to the VCP. Upper: Cartoon representing the predicted VCPstructure using the mouse crystal structure of p97 (PDB ID 3CF1); sticksrepresent the structure of HV-3. Lower: the enlarged area. (E) Thesequence in VCP corresponding to HV-3 in Htt was deleted (AVCP). GST,GST-VCP, or GST-AVCP was incubated with total lysates of mouse brain for16 hours followed by immunoblotting (IB) with anti-Htt antibodies.

FIG. 9 illustrates plots showing HV-3 dose response in animal models ofR6/2 mice.

DETAILED DESCRIPTION

The embodiments described herein are not limited to the particularmethodology, protocols, and reagents, etc., and as such may vary. Theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which is defined solely by the claims. Other than in theoperating examples, or where otherwise indicated, all numbers expressingquantities of ingredients or reaction conditions used herein should beunderstood as modified in all instances by the term “about.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments are based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Unless otherwise defined, scientific and technical terms used hereinshall have the meanings that are commonly understood by those ofordinary skill in the art. Further, unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. Generally, nomenclatures utilized in connectionwith, and techniques of, cell and tissue culture, molecular biology, andprotein and oligo- or polynucleotide chemistry and hybridizationdescribed herein are those well known and commonly used in the art.

As used herein, “one or more of a, b, and c” means a, b, c, ab, ac, bc,or abc. The use of “or” herein is the inclusive or.

The term “administering” to a patient includes dispensing, delivering orapplying an active compound in a pharmaceutical formulation to a subjectby any suitable route for delivery of the active compound to the desiredlocation in the subject (e.g., to thereby contact a desired cell such asa desired neuron), including administration into the cerebrospinal fluidor across the blood-brain barrier, delivery by either the parenteral ororal route, intramuscular injection, subcutaneous or intradermalinjection, intravenous injection, buccal administration, transdermaldelivery and administration by the rectal, colonic, vaginal, intranasalor respiratory tract route. The agents may, for example, be administeredto a comatose, anesthetized or paralyzed subject via an intravenousinjection or may be administered intravenously to a pregnant subject tostimulate axonal growth in a fetus. Specific routes of administrationmay include topical application (such as by eyedrops, creams or erodibleformulations to be placed under the eyelid, intraocular injection intothe aqueous or the vitreous humor, injection into the external layers ofthe eye, such as via subconjunctival injection or subtenon injection,parenteral administration or via oral routes.

The term “antibody”, includes human and animal mAbs, and preparations ofpolyclonal antibodies, synthetic antibodies, including recombinantantibodies (antisera), chimeric antibodies, including humanizedantibodies, anti-idiotopic antibodies and derivatives thereof. A portionor fragment of an antibody refers to a region of an antibody thatretains at least part of its ability (binding specificity and affinity)to bind to a specified epitope. The term “epitope” or “antigenicdeterminant” refers to a site on an antigen to which antibody paratopebinds. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, at least 5, or 8 to10, or about 13 to 15 amino acids in a unique spatial conformation.Methods of determining spatial conformation of epitopes include, forexample, x-ray crystallography and 2-dimensional nuclear magneticresonance.

The terms “chimeric protein” or “fusion protein” refer to a fusion of afirst amino acid sequence encoding a polypeptide with a second aminoacid sequence defining a domain (e g, polypeptide portion) foreign toand not substantially homologous with the domain of the firstpolypeptide. A chimeric protein may present a foreign domain, which isfound (albeit in a different protein) in an organism, which alsoexpresses the first protein, or it may be an “interspecies”,“intergenic”, etc. fusion of protein structures expressed by differentkinds of organisms.

The term “contacting nerves”, “contacting neurons”, “treating nerves”,or “treating neurons” refers to any mode of agent delivery or“administration,” either to cells or to whole organisms, in which theagent is capable of exhibiting its pharmacological effect in neurons.“Contacting neurons” includes both in vivo and in vitro methods ofbringing an agent of the invention into proximity with a neuron.Suitable modes of administration can be determined by those skilled inthe art and such modes of administration may vary between agents.

An “effective amount” of an agent or therapeutic peptide is an amountsufficient to achieve a desired therapeutic or pharmacological effect,such as an amount that is capable of activating the growth of neurons.An effective amount of an agent as defined herein may vary according tofactors such as the disease state, age, and weight of the subject, andthe ability of the agent to elicit a desired response in the subject.Dosage regimens may be adjusted to provide the optimum therapeuticresponse. An effective amount is also one in which any toxic ordetrimental effects of the active compound are outweighed by thetherapeutically beneficial effects.

The term “expression” refers to the process by which nucleic acid istranslated into peptides or is transcribed into RNA, which, for example,can be translated into peptides, polypeptides or proteins. If thenucleic acid is derived from genomic DNA, expression may, if anappropriate eukaryotic host cell or organism is selected, includesplicing of the mRNA. For heterologous nucleic acid to be expressed in ahost cell, it must initially be delivered into the cell and then, oncein the cell, ultimately reside in the nucleus.

The term “genetic therapy” involves the transfer of heterologous DNA tocells of a mammal, particularly a human, with a disorder or conditionsfor which therapy or diagnosis is sought. The DNA is introduced into theselected target cells in a manner such that the heterologous DNA isexpressed and a therapeutic product encoded thereby is produced.Alternatively, the heterologous DNA may in some manner mediateexpression of DNA that encodes the therapeutic product; it may encode aproduct, such as a peptide or RNA that in some manner mediates, directlyor indirectly, expression of a therapeutic product. Genetic therapy mayalso be used to deliver nucleic acid encoding a gene product to replacea defective gene or supplement a gene product produced by the mammal orthe cell in which it is introduced. The introduced nucleic acid mayencode a therapeutic compound, such as a growth factor inhibitorthereof, or a tumor necrosis factor or inhibitor thereof, such as areceptor therefore, that is not normally produced in the mammalian hostor that is not produced in therapeutically effective amounts or at atherapeutically useful time. The heterologous DNA encoding thetherapeutic product may be modified prior to introduction into the cellsof the afflicted host in order to enhance or otherwise alter the productor expression thereof.

The term “gene” or “recombinant gene” refers to a nucleic acidcomprising an open reading frame encoding a polypeptide, including bothexon and (optionally) intron sequences.

The term “heterologous nucleic acid sequence” is typically DNA thatencodes RNA and proteins that are not normally produced in vivo by thecell in which it is expressed or that mediates or encodes mediators thatalter expression of endogenous DNA by affecting transcription,translation, or other regulatable biochemical processes. A heterologousnucleic acid sequence may also be referred to as foreign DNA. Any DNAthat one of skill in the art would recognize or consider as heterologousor foreign to the cell in which it is expressed is herein encompassed byheterologous DNA. Examples of heterologous DNA include, but are notlimited to, DNA that encodes traceable marker proteins, such as aprotein that confers drug resistance, DNA that encodes therapeuticallyeffective substances, such as anti-cancer agents, enzymes and hormones,and DNA that encodes other types of proteins, such as antibodies.Antibodies that are encoded by heterologous DNA may be secreted orexpressed on the surface of the cell in which the heterologous DNA hasbeen introduced.

The terms “homology” and “identity” are used synonymously throughout andrefer to sequence similarity between two peptides or between two nucleicacid molecules. Homology can be determined by comparing a position ineach sequence, which may be aligned for purposes of comparison. When aposition in the compared sequence is occupied by the same base or aminoacid, then the molecules are homologous or identical at that position. Adegree of homology or identity between sequences is a function of thenumber of matching or homologous positions shared by the sequences.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrastemalinjection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into a target tissue, such that it enters theanimal's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

The term “patient” or “subject” or “animal” or “host” refers to anymammal. The subject may be a human, but can also be a mammal in need ofveterinary treatment, e.g., domestic animals (e.g., dogs, cats, and thelike), farm animals (e.g., cows, sheep, fowl, pigs, horses, and thelike) and laboratory animals (e.g., rats, mice, guinea pigs, and thelike).

The terms “polynucleotide sequence” and “nucleotide sequence” are alsoused interchangeably herein.

The terms “peptide” or “polypeptide” are used interchangeably herein andrefer to compounds consisting of from about 2 to about 90 amino acidresidues, inclusive, wherein the amino group of one amino acid is linkedto the carboxyl group of another amino acid by a peptide bond. A peptidecan be, for example, derived or removed from a native protein byenzymatic or chemical cleavage, or can be prepared using conventionalpeptide synthesis techniques (e.g., solid phase synthesis) or molecularbiology techniques (see Sambrook et al., MOLECULAR CLONING: LAB. MANUAL(Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989)). A “peptide”can comprise any suitable L- and/or D-amino acid, for example, commona-amino acids (e.g., alanine, glycine, valine), non-a-amino acids (e.g.,P-alanine, 4-aminobutyric acid, 6aminocaproic acid, sarcosine, statine),and unusual amino acids (e.g., citrulline, homocitruline, homoserine,norleucine, norvaline, ornithine). The amino, carboxyl and/or otherfunctional groups on a peptide can be free (e g, unmodified) orprotected with a suitable protecting group. Suitable protecting groupsfor amino and carboxyl groups, and means for adding or removingprotecting groups are known in the art. See, e.g., Green &amp; Wuts,PROTECTING GROUPS IN ORGANIC SYNTHESIS (John Wiley &amp; Sons, 1991).The functional groups of a peptide can also be derivatized (e.g.,alkylated) using art-known methods.

The term “peptidomimetic”, refers to a protein-like molecule designed tomimic a peptide. Peptidomimetics typically arise either frommodification of an existing peptide, or by designing similar systemsthat mimic peptides, such as peptoids and β-peptides. Irrespective ofthe approach, the altered chemical structure is designed toadvantageously adjust the molecular properties such as, stability orbiological activity. These modifications involve changes to the peptidethat do not occur naturally (such as altered backbones and theincorporation of nonnatural amino acids).

The terms “prevent” or “preventing” refer to reducing the frequency orseverity of a disease or condition. The term does not require anabsolute preclusion of the disease or condition. Rather, this termincludes decreasing the chance for disease occurrence.

A polynucleotide sequence (DNA, RNA) is “operatively linked” to anexpression control sequence when the expression control sequencecontrols and regulates the transcription and translation of thatpolynucleotide sequence. The term “operatively linked” includes havingan appropriate start signal (e.g., ATG) in front of the polynucleotidesequence to be expressed, and maintaining the correct reading frame topermit expression of the polynucleotide sequence under the control ofthe expression control sequence, and production of the desiredpolypeptide encoded by the polynucleotide sequence.

The term “recombinant,” as used herein, means that a protein is derivedfrom a prokaryotic or eukaryotic expression system.

The term “therapeutically effective” means that the amount of thecomposition used is of sufficient quantity to ameliorate one or morecauses, symptoms, or sequelae of a disease or disorder. Suchamelioration only requires a reduction or alteration, not necessarilyelimination, of the causes, symptoms, or sequelae of a disease ordisorder.

The term “tissue-specific promoter” means a nucleic acid sequence thatserves as a promoter, i.e., regulates expression of a selected nucleicacid sequence operably linked to the promoter, and which affectsexpression of the selected nucleic acid sequence in specific cells of atissue, such as cells of epithelial cells. The term also coversso-called “leaky” promoters, which regulate expression of a selectednucleic acid primarily in one tissue, but cause expression in othertissues as well. The term “transfection” is used to refer to the uptakeof foreign DNA by a cell. A cell has been “transfected” when exogenousDNA has been introduced inside the cell membrane. A number oftransfection techniques are generally known in the art. See, e.g.,Graham et al., Virology 52:456 (1973); Sambrook et al., MolecularCloning: A Laboratory Manual (1989); Davis et al., Basic Methods inMolecular Biology (1986); Chu et al., Gene 13:197 (1981). Suchtechniques can be used to introduce one or more exogenous DNA moieties,such as a nucleotide integration vector and other nucleic acidmolecules, into suitable host cells. The term captures chemical,electrical, and viral-mediated transfection procedures.

The terms “transcriptional regulatory sequence” is a generic term usedthroughout the specification to refer to nucleic acid sequences, such asinitiation signals, enhancers, and promoters, which induce or controltranscription of protein coding sequences with which they are operablylinked. In some examples, transcription of a recombinant gene is underthe control of a promoter sequence (or other transcriptional regulatorysequence), which controls the expression of the recombinant gene in acell-type in which expression is intended. It will also be understoodthat the recombinant gene can be under the control of transcriptionalregulatory sequences which are the same or which are different fromthose sequences, which control transcription of the naturally occurringform of a protein.

The term “treatment” refers to the medical management of a patient withthe intent to cure, ameliorate, stabilize, or prevent a disease,pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. Preferredvectors are those capable of one or more of, autonomous replication andexpression of nucleic acids to which they are linked. Vectors capable ofdirecting the expression of genes to which they are operatively linkedare referred to herein as “expression vectors”.

The term “wild type” refers to the naturally-occurring polynucleotidesequence encoding a protein, or a portion thereof, or protein sequence,or portion thereof, respectively, as it normally exists in vivo. As usedherein, the term “nucleic acid” refers to polynucleotides, such asdeoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

The agents, compounds, compositions, antibodies, etc. used in themethods described herein are considered to be purified and/or isolatedprior to their use. Purified materials are typically “substantiallypure”, meaning that a nucleic acid, polypeptide or fragment thereof, orother molecule has been separated from the components that naturallyaccompany it. Typically, the polypeptide is substantially pure when itis at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free fromthe proteins and other organic molecules with which it is associatednaturally. For example, a substantially pure polypeptide may be obtainedby extraction from a natural source, by expression of a recombinantnucleic acid in a cell that does not normally express that protein, orby chemical synthesis. “Isolated materials” have been removed from theirnatural location and environment. In the case of an isolated or purifieddomain or protein fragment, the domain or fragment is substantially freefrom amino acid sequences that flank the protein in thenaturally-occurring sequence. The term “isolated DNA” means DNA has beensubstantially freed of the genes that flank the given DNA in thenaturally occurring genome. Thus, the term “isolated DNA” encompasses,for example, cDNA, cloned genomic DNA, and synthetic DNA.

The terms “portion”, “fragment”, “variant”, “derivative” and “analog”,when referring to a polypeptide include any polypeptide that retains atleast some biological activity referred to herein (e.g., inhibition ofan interaction such as binding). Polypeptides as described herein mayinclude portion, fragment, variant, or derivative molecules withoutlimitation, as long as the polypeptide still serves its function.Polypeptides or portions thereof of the present invention may includeproteolytic fragments, deletion fragments and in particular, orfragments that more easily reach the site of action when delivered to ananimal.

Embodiments described herein relate to compositions and methods forinhibiting aberrant Valosin-Containing Protein (VCP) accumulation in themitochondria, and particularly relates to compositions and methods fortreating disease or disorders associated with aberrant VCP accumulationin the mitochondria of nerve cells.

VCP, also known as p97 in vertebrates and Cdc48 in S. cerevisiae, is aclass II member of the ATPase associated with diverse cellularactivities. VCP is highly conserved from archaebacteria to humans, andis located in different subcellular organelles, including theendoplasmic reticulum (ER), mitochondria, and nucleus, where itfunctions in diverse cellular processes including ER-associated proteindegradation (ERAD), mitochondria-associated degradation (MAD),autophagy, and DNA repair. VCP can translocate to mitochondria, where itis required for turnover of mitochondrial outer membrane proteinMitofusins and parkin-dependent mitophagy. Overexpression of VCP resultsin mitochondrial fragmentation and cell death in neurons exposed tovarious mitochondrial toxins, such as rotenone, 6-OHDA, Aβ-peptides.Pathogenic mutations in the VCP in yeast and Drosophila causemitochondrial depolarization, mitochondrial oxidative stress, reducedATP production, and mitochondrial aggregations. Mice with VCP mutantsdisplayed degeneration of mitochondria, enhanced autophagy, motor neurondegeneration, and early lethality. Mutations of VCP gene in humans causefrontotemporal dementia, amyotrophic lateral sclerosis (ALS), andmuscular and bone degeneration, all of which are manifestations ofmitochondrial dysfunction.

VCP was found to be involved in the pathogenesis of polyglutamine(polyQ) diseases. First, endogenous VCP was found to co-localize withpolyglutamine-containing aggregates that were found in patients with HDand Machado-Joseph disease. Second, VCP can directly bind to multiplepolyglutamine disease proteins, including huntingtin, ataxin-1,ataxin-7, and androgen receptors. Third, in a transgenic Drosophilamodel expressing a fragments of polyQ gene carrying either 79 or 92 CAGrepeats, an up-regulation of VCP expression was observed prior to celldeath, and over-expression of VCP severely enhanced eye degeneration.Thus, VCP can act as a cell death effector in polyQ-inducedneurodegeneration.

VCP was found to be aberrantly recruited to mitochondria viamitochondria-bound mutant huntingtin protein (mHtt) protein-proteininteraction. Wild-type huntingtin protein (Htt) can serve as a scaffoldprotein that regulates various physiological processes throughprotein-protein interactions. VCP was found to bind to mtHtt in HDbrains, and endogenous VCP can co-localize with mtHtt aggregates in thecytoplasm in primary neurons expressing a fragment of mtHtt. Suchaccumulation of VCP on mitochondria resulted in excessive mitophagy,mitochondrial mass loss, and subsequent neuronal degeneration in HDmodels in culture and in animals.

It was found that blocking VCP translocation to mitochondria byinhibiting VCP/mtHtt protein interactions inhibited VCP-mediatedmitophagy impairment, suppressed mitochondrial dysfunction, and reducedHD-associated neuropathology and motor deficits in two HD transgenicmouse models and thus can be used to treat neurodegneration associatedwith aberrant accumulation of VCP in the mitochondria. Indeed, in vitroand in vivo data described herein shows that blocking VCP/mtHtt bindingabolished VCP translocation to the mitochondria and reducedmitochondrial damage, further emphasizing that the binding of VCP/mtHttis required for VCP relocation to the mitochondria. Significantly,inhibition of VCP/mtHtt binding reduced HD-related behavioral andpathological phenotypes in two HD transgenic mice. Thus, the formationof aberrant complex of VCP/mtHtt on the mitochondria is a key step ininitiating mitochondrial injury, which in turn results in neuronalpathology in HD. Inhibitors that selectively block VCP mitochondrialaccumulation can provide a therapeutic route for HD and multiplepolyglutamine (polyQ) diseases in which the binding of VCP with mutantpolyQ proteins and associated mitochondrial defects are characterized.

Accordingly, therapeutic agents that inhibit aberrant VCP accumulationin the mitochondria by polyQ proteins can be use in methods of treatingpolyQ mediated neurodegeneration and/or neurodegenerative diseases in asubject in need thereof. The polyQ mediated neurodegeneration and/orneurodegenerative diseases can include, for example, Huntington'sdisease, spinocerebellar ataxias of types 1, 2, 3, 6, 7 and 17,dentatorubral pallidoluysian atrophy as well as spinobulbar muscularatrophy (Kennedy syndrome).

Other neudegenerative diseases that can also be treated by therapeuticagents that inhibit aberrant VCP accumulation in the mitochondria caninclude Parkinson's disease, Multiple Sclerosis (MS), AmyotrophicLateral Sclerosis (ALS), multi-system atrophy, Alzheimer's disease,stroke, Spinal-cerebellar ataxia, progressive supranuclear palsy,progressive supernuclear palsy, granulovacuolar disease, frontotemporaldementia, corticobasal degeneration, epilepsy, autoimmuneencephalomyelitis, diabetic neuropathy, glaucomatous neuropathy, Pick'sdisease, Lewy Body disease, Creutzfeld-Jacob Disease (CJD), variantCreutzfeld-Jacob Disease, new variant Creutzfeld-Jacob Disease, or kurudisease.

In some embodiments, a therapeutic agent that inhibits or reducesaberrant VCP accumulation in the mitochondria by polyQ proteins, caninclude a therapeutic peptide or small molecule that binds to and/orcomplexes with VCP to inhibit the binding or complexing of VCP and thepolyQ protein, such as mtHtt. Accordingly, therapeutic peptides or smallmolecules that bind to and/or complex with VCP to inhibit or reducebinding or complexing of VCP and the polyQ protein as well as inhibitaberrant VCP accumulation in the mitochondria can be used to treatneurodegerative diseases or disorders, such as Huntington's disease, ina subject in need thereof.

In some embodiments, the therapeutic agent can comprise a short peptidederived from interaction sites between VCP and the polyQ protein thatinhibits or reduces binding or complexing of VCP and the polyQ protein.For example, the therapeutic agent can comprise short peptides of VCP ormtHtt that can inhibit the interaction of VCP and mtHtt. In oneembodiment, the therapeutic agent can comprise a short peptide that isderived from Htt and represents a sequence homologous to VCP. Thetherapeutic peptide can have at least about 75% sequence identity toabout 8 to about 10 consecutive amino acids of an interaction site ofVCP with the polyglutamine protein. In some embodiments, the therapeuticpeptide can have an amino acid sequence that is at least about 75%identical to SEQ ID NO: 4. For example, the therapeutic peptide can havean amino acid sequence of SEQ ID NO: 3. A short peptide having the aminoacid of SEQ ID NO: 3 is referred herein as HV-3. HV-3 was found to blockthe binding of VCP to mtHtt in cultures and in animal models of HD,which shows that HV-3 can compete with Htt binding to VCP and/or that itcan prevent the exposure of the VCP-binding site on Htt.

In some embodiments, the therapeutic agent or therapeutic peptide caninclude, a peptide that consists essentially, and/or consists of about 8to about 12 amino acids and has an amino acid sequence that is at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, or about100% homologous to an about 6 to about 12 consecutive amino acids (e.g.,about 8 to about 10 consecutive amino acids) of an interaction site ofVCP and polyQ protein, such as a petide having the amino acid sequenceof SEQ ID NO: 3 or SEQ ID NO: 4.

The therapeutic peptides described herein can be subject to othervarious changes, substitutions, insertions, and deletions where suchchanges provide for certain advantages in its use. In this regard,therapeutic peptides that bind to and/or complex with an interactionsite of VCP and a polyQ protein can correspond to or be substantiallyhomologous with, rather than be identical to, the sequence of a recitedpolypeptide where one or more changes are made and it retains theability to inhibits or reduces interaction of VCP and a polyQ protein toinhibit aberrant VCP accumulation in the mitochondrial of a cell, suchas a neuron.

The therapeutic peptide can be in any of a variety of forms ofpolypeptide derivatives that include amides, conjugates with proteins,cyclized polypeptides, polymerized polypeptides, analogs, fragments,chemically modified polypeptides and the like derivatives.

It will be appreciated that the conservative substitution can alsoinclude the use of a chemically derivatized residue in place of anon-derivatized residue provided that such peptide displays therequisite binding activity.

“Chemical derivative” refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized molecules include for example, those molecules in whichfree amino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups maybe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included as chemicalderivatives are those polypeptides, which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexamples: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine. Polypeptides described hereinalso include any polypeptide having one or more additions and/ordeletions or residues relative to the sequence of a polypeptide whosesequence is shown herein, so long as the requisite activity ismaintained.

One or more of peptides of the therapeutic peptides described herein canalso be modified by natural processes, such as posttranslationalprocessing, and/or by chemical modification techniques, which are knownin the art. Modifications may occur in the peptide including the peptidebackbone, the amino acid side-chains and the amino or carboxy termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given peptide.Modifications comprise for example, without limitation, acetylation,acylation, addition of acetomidomethyl (Acm) group, ADP-ribosylation,amidation, covalent attachment to fiavin, covalent attachment to a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphatidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation and ubiquitination (for reference see, Protein-structureand molecular properties, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New-York, 1993).

Peptides and/or proteins described herein may also include, for example,biologically active mutants, variants, fragments, chimeras, andanalogues; fragments encompass amino acid sequences having truncationsof one or more amino acids, wherein the truncation may originate fromthe amino terminus (N-terminus), carboxy terminus (C-terminus), or fromthe interior of the protein. Analogues of the invention involve aninsertion or a substitution of one or more amino acids. Variants,mutants, fragments, chimeras and analogues may function as inhibitors ofthe interaction of VCP and polyQ proteins (without being restricted tothe present examples).

The therapeutic peptides described herein may be prepared by methodsknown to those skilled in the art. The peptides and/or proteins may beprepared using recombinant DNA. For example, one preparation can includecultivating a host cell (bacterial or eukaryotic) under conditions,which provide for the expression of peptides and/or proteins within thecell.

The purification of the polypeptides may be done by affinity methods,ion exchange chromatography, size exclusion chromatography,hydrophobicity or other purification technique typically used forprotein purification. The purification step can be performed undernon-denaturating conditions. On the other hand, if a denaturating stepis required, the protein may be renatured using techniques known in theart.

In some embodiments, the therapeutic peptides described herein caninclude additional residues that may be added at either terminus of apolypeptide for the purpose of providing a “linker” by which thepolypeptides can be conveniently linked and/or affixed to otherpolypeptides, proteins, detectable moieties, labels, solid matrices, orcarriers.

Amino acid residue linkers are usually at least one residue and can be40 or more residues, more often 1 to 10 residues. Typical amino acidresidues used for linking are glycine, tyrosine, cysteine, lysine,glutamic and aspartic acid, or the like. In addition, a subjectpolypeptide can differ by the sequence being modified by terminal-NH2acylation, e.g., acetylation, or thioglycolic acid amidation, byterminal-carboxylamidation, e.g., with ammonia, methylamine, and thelike terminal modifications. Terminal modifications are useful, as iswell known, to reduce susceptibility by proteinase digestion, andtherefore serve to prolong half life of the polypeptides in solutions,particularly biological fluids where proteases may be present. In thisregard, polypeptide cyclization is also a useful terminal modification,and is particularly preferred also because of the stable structuresformed by cyclization and in view of the biological activities observedfor such cyclic peptides as described herein.

In some embodiments, the linker can be a flexible peptide linker thatlinks the therapeutic peptide to other polypeptides, proteins, and/ormolecules, such as detectable moieties, labels, solid matrices, orcarriers. A flexible peptide linker can be about 20 or fewer amino acidsin length. For example, a peptide linker can contain about 12 or feweramino acid residues, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In somecases, a peptide linker comprises two or more of the following aminoacids: glycine, serine, alanine, and threonine.

In some embodiments, a therapeutic agent comprising the therapeuticpeptides described herein can be provided in the form of a conjugateprotein or drug delivery construct includes at least a transportsubdomain(s) or moiety(ies) (i.e., transport moieties or cellpenetrating moieties) that is linked to the therapeutic peptide. Thetransport moieties can facilitate uptake of the therapeutic polypeptidesinto a mammalian (i e, human or animal) tissue or cell (e.g., neuralcell). The transport moieties can be covalently linked to thetherapeutic polypeptides. The covalent link can include a peptide bondor a labile bond (e.g., a bond readily cleavable or subject to chemicalchange in the interior target cell environment). Additionally, thetransport moieties can be cross-linked (e.g., chemically cross-linked,UV cross-linked) to the therapeutic polypeptide. The transport moietiescan also be linked to the therapeutic polypeptide with linkingpolypeptide described herein.

The transport moieties can be repeated more than once in the therapeuticagent. The repetition of a transport moiety may affect (e.g., increase)the uptake of the peptides and/or proteins by a desired cell. Thetransport moiety may also be located either at the amino-terminal regionof therapeutic peptide or at its carboxy-terminal region or at bothregions.

In one embodiment, the transport moiety can include at least onetransport peptide sequence that allows the therapeutic polypeptide oncelinked to the transport moiety to penetrate into the cell by areceptor-independent mechanism. In one example, the transport peptide isa synthetic peptide that contains a Tat-mediated protein deliverysequence and SEQ ID NO: 3.

Other examples of known transport moieties, subdomains and the like aredescribed in, for example, Canadian patent document No. 2,301,157(conjugates containing homeodomain of antennapedia) as well as in U.S.Pat. Nos. 5,652,122, 5,670,617, 5,674,980, 5,747,641, and 5,804,604, allof which are incorporated herein by reference in their entirety,(conjugates containing amino acids of Tat HIV protein; herpes simplexvirus-1 DNA binding protein VP22, a Histidine tag ranging in length from4 to 30 histidine repeats, or a variation derivative or homologuethereof capable of facilitating uptake of the active cargo moiety by areceptor independent process.

A 16 amino acid region of the third alpha-helix of antennapediahomeodomain has also been shown to enable proteins (made as fusionproteins) to cross cellular membranes (PCT international publicationnumber WO 99/11809 and Canadian application No.: 2,301,157. Similarly,HIV Tat protein was shown to be able to cross cellular membranes.

In addition, the transport moiety(ies) can include polypeptides having abasic amino acid rich region covalently linked to an active agent moiety(e.g., intracellular domain-containing fragments inhibitor peptide). Asused herein, the term “basic amino acid rich region” relates to a regionof a protein with a high content of the basic amino acids such asarginine, histidine, asparagine, glutamine, lysine. A “basic amino acidrich region” may have, for example 15% or more of basic amino acid. Insome instance, a “basic amino acid rich region” may have less than 15%of basic amino acids and still function as a transport agent region. Inother instances, a basic amino acid region will have 30% or more ofbasic amino acids.

The transport moiety(ies) may further include a proline rich region. Asused herein, the term proline rich region refers to a region of apolypeptide with 5% or more (up to 100%) of proline in its sequence. Insome instance, a proline rich region may have between 5% and 15% ofprolines. Additionally, a proline rich region refers to a region, of apolypeptide containing more prolines than what is generally observed innaturally occurring proteins (e.g., proteins encoded by the humangenome). Proline rich regions of this application can function as atransport agent region.

In one embodiment, the therapeutic peptide described herein can benon-covalently linked to a transduction agent. An example of anon-covalently linked polypeptide transduction agent is the Chariotprotein delivery system (See U.S. Pat. No. 6,841,535; J Biol Chem274(35):24941-24946; and Nature Biotec. 19:1173-1176, all hereinincorporated by reference in their entirety).

In other embodiments, the therapeutic peptides can be expressed in cellsbeing treated using gene therapy to inhibit VCP interaction or bindingwith the polyQ protein, such as mtHtt. The gene therapy can use a vectorincluding a nucleotide encoding the therapeutic peptides. A “vector”(sometimes referred to as gene delivery or gene transfer “vehicle”)refers to a macromolecule or complex of molecules comprising apolynucleotide to be delivered to the cell. The polynucleotide to bedelivered may comprise a coding sequence of interest in gene therapy.Vectors include, for example, viral vectors (such as adenoviruses (Ad),adeno-associated viruses (AAV), and retroviruses), liposomes and otherlipid-containing complexes, and other macromolecular complexes capableof mediating delivery of a polynucleotide to a target cell.

Vectors can also comprise other components or functionalities thatfurther modulate gene delivery and/or gene expression, or that otherwiseprovide beneficial properties to the targeted cells. Such othercomponents include, for example, components that influence binding ortargeting to cells (including components that mediate cell-type ortissue-specific binding); components that influence uptake of the vectornucleic acid by the cell; components that influence localization of thepolynucleotide within the cell after uptake (such as agents mediatingnuclear localization); and components that influence expression of thepolynucleotide. Such components also might include markers, such asdetectable and/or selectable markers that can be used to detect orselect for cells that have taken up and are expressing the nucleic aciddelivered by the vector. Such components can be provided as a naturalfeature of the vector (such as the use of certain viral vectors whichhave components or functionalities mediating binding and uptake), orvectors can be modified to provide such functionalities.

Selectable markers can be positive, negative or bifunctional. Positiveselectable markers allow selection for cells carrying the marker,whereas negative selectable markers allow cells carrying the marker tobe selectively eliminated. A variety of such marker genes have beendescribed, including bifunctional (i.e., positive/negative) markers(see, e.g., Lupton, S., WO 92/08796, published May 29, 1992; and Lupton,S., WO 94/28143, published Dec. 8, 1994). Such marker genes can providean added measure of control that can be advantageous in gene therapycontexts. A large variety of such vectors are known in the art and aregenerally available.

Vectors for use herein include viral vectors, lipid based vectors andother non-viral vectors that are capable of delivering a nucleotideencoding the therapeutic peptides described herein to the target cells.The vector can be a targeted vector, especially a targeted vector thatpreferentially binds to neurons and. Viral vectors for use in theapplication can include those that exhibit low toxicity to a target celland induce production of therapeutically useful quantities of thetherapeutic peptide in a cell specific manner.

Examples of viral vectors are those derived from adenovirus (Ad) oradeno-associated virus (AAV). Both human and non-human viral vectors canbe used and the recombinant viral vector can be replication-defective inhumans. Where the vector is an adenovirus, the vector can comprise apolynucleotide having a promoter operably linked to a gene encoding thetherapeutic peptides and is replication-defective in humans.

Other viral vectors that can be used herein include herpes simplex virus(HSV)-based vectors. HSV vectors deleted of one or more immediate earlygenes (IE) are advantageous because they are generally non-cytotoxic,persist in a state similar to latency in the target cell, and affordefficient target cell transduction. Recombinant HSV vectors canincorporate approximately 30 kb of heterologous nucleic acid.

Retroviruses, such as C-type retroviruses and lentiviruses, might alsobe used in the application. For example, retroviral vectors may be basedon murine leukemia virus (MLV). See, e.g., Hu and Pathak, Pharmacol.Rev. 52:493-511, 2000 and Fong et al., Crit. Rev. Ther. Drug CarrierSyst. 17:1-60, 2000. MLV-based vectors may contain up to 8 kb ofheterologous (therapeutic) DNA in place of the viral genes. Theheterologous DNA may include a tissue-specific promoter and a nucleicacid encoding the therapeutic peptide. In methods of delivery to neuralcells, it may also encode a ligand to a tissue specific receptor.

Additional retroviral vectors that might be used arereplication-defective lentivirus-based vectors, including humanimmunodeficiency (HIV)-based vectors. See, e.g., Vigna and Naldini, J.Gene Med. 5:308-316, 2000 and Miyoshi et al., J. Virol. 72:8150-8157,1998. Lentiviral vectors are advantageous in that they are capable ofinfecting both actively dividing and non-dividing cells.

Lentiviral vectors for use in the application may be derived from humanand non-human (including SIV) lentiviruses. Examples of lentiviralvectors include nucleic acid sequences required for vector propagationas well as a tissue-specific promoter operably linked to a therapeuticpeptide encoding nucleic acid. These former may include the viral LTRs,a primer binding site, a polypurine tract, att sites, and anencapsidation site.

In some aspects, a lentiviral vector can be employed. Lentiviruses haveproven capable of transducing different types of CNS neurons (Azzouz etal., (2002) J Neurosci. 22: 10302-12) and may be used in someembodiments because of their large cloning capacity.

A lentiviral vector may be packaged into any lentiviral capsid. Thesubstitution of one particle protein with another from a different virusis referred to as “pseudotyping”. The vector capsid may contain viralenvelope proteins from other viruses, including murine leukemia virus(MLV) or vesicular stomatitis virus (VSV). The use of the VSV G-proteinyields a high vector titer and results in greater stability of thevector virus particles.

Alphavirus-based vectors, such as those made from semliki forest virus(SFV) and sindbis virus (SIN) might also be used in the application. Useof alphaviruses is described in Lundstrom, K., Intervirology 43:247-257,2000 and Perri et al., Journal of Virology 74:9802-9807, 2000.

Recombinant, replication-defective alphavirus vectors are advantageousbecause they are capable of high-level heterologous (therapeutic) geneexpression, and can infect a wide target cell range. Alphavirusreplicons may be targeted to specific cell types by displaying on theirvirion surface a functional heterologous ligand or binding domain thatwould allow selective binding to target cells expressing a cognatebinding partner. Alphavirus replicons may establish latency, andtherefore long-term heterologous nucleic acid expression in a targetcell. The replicons may also exhibit transient heterologous nucleic acidexpression in the target cell.

In many of the viral vectors compatible with methods of the application,more than one promoter can be included in the vector to allow more thanone heterologous gene to be expressed by the vector. Further, the vectorcan comprise a sequence, which encodes a signal peptide or other moiety,which facilitates expression of the therapeutic peptide from the targetcell.

To combine advantageous properties of two viral vector systems, hybridviral vectors may be used to deliver a nucleic acid encoding atherapeutic peptide to a target neuron, cell, or tissue. Standardtechniques for the construction of hybrid vectors are well-known tothose skilled in the art. Such techniques can be found, for example, inSambrook, et al., In Molecular Cloning: A laboratory manual. Cold SpringHarbor, N.Y. or any number of laboratory manuals that discussrecombinant DNA technology. Double-stranded AAV genomes in adenoviralcapsids containing a combination of AAV and adenoviral ITRs may be usedto transduce cells. In another variation, an AAV vector may be placedinto a “gutless”, “helper-dependent” or “high-capacity” adenoviralvector. Adenovirus/AAV hybrid vectors are discussed in Lieber et al., J.Virol. 73:9314-9324, 1999. Retrovirus/adenovirus hybrid vectors arediscussed in Zheng et al., Nature Biotechnol. 18:176-186, 2000.Retroviral genomes contained within an adenovirus may integrate withinthe target cell genome and effect stable gene expression.

Other nucleotide sequence elements, which facilitate expression of thetherapeutic peptide and cloning of the vector are further contemplated.For example, the presence of enhancers upstream of the promoter orterminators downstream of the coding region, for example, can facilitateexpression.

In accordance with another embodiment, a tissue-specific promoter can befused to nucleotides encoding the therapeutic peptides described herein.By fusing such tissue specific promoter within the adenoviral construct,transgene expression is limited to a particular tissue. The efficacy ofgene expression and degree of specificity provided by tissue specificpromoters can be determined, using the recombinant adenoviral system ofthe present application. Neuron specific promoters, such as theplatelet-derived growth factor β-chain (PDGF-β) promoter and vectors,are well known in the art.

In addition to viral vector-based methods, non-viral methods may also beused to introduce a nucleic acid encoding a therapeutic peptide into atarget cell. A review of non-viral methods of gene delivery is providedin Nishikawa and Huang, Human Gene Ther. 12:861-870, 2001. An example ofa non-viral gene delivery method according to the application employsplasmid DNA to introduce a nucleic acid encoding a therapeutic peptideinto a cell. Plasmid-based gene delivery methods are generally known inthe art.

Synthetic gene transfer molecules can be designed to form multimolecularaggregates with plasmid DNA. These aggregates can be designed to bind toa target cell. Cationic amphiphiles, including lipopolyamines andcationic lipids, may be used to provide receptor-independent nucleicacid transfer into target cells.

In addition, preformed cationic liposomes or cationic lipids may bemixed with plasmid DNA to generate cell-transfecting complexes. Methodsinvolving cationic lipid formulations are reviewed in Feigner et al.,Ann. N.Y. Acad. Sci. 772:126-139, 1995 and Lasic and Templeton, Adv.Drug Delivery Rev. 20:221-266, 1996. For gene delivery, DNA may also becoupled to an amphipathic cationic peptide (Fominaya et al., J. GeneMed. 2:455-464, 2000).

Methods that involve both viral and non-viral based components may beused according to the application. For example, an Epstein Barr virus(EBV)-based plasmid for therapeutic gene delivery is described in Cui etal., Gene Therapy 8:1508-1513, 2001. Additionally, a method involving aDNA/ligand/polycationic adjunct coupled to an adenovirus is described inCuriel, D. T., Nat. Immun 13:141-164, 1994.

Additionally, the nucleic acid encoding the therapeutic peptides can beintroduced into the target cell by transfecting the target cells usingelectroporation techniques. Electroporation techniques are well knownand can be used to facilitate transfection of cells using plasmid DNA.

Vectors that encode the expression of the therapeutic peptides can bedelivered in vivo to the target cell in the form of an injectablepreparation containing pharmaceutically acceptable carrier, such assaline, as necessary. Other pharmaceutical carriers, formulations anddosages can also be used in accordance with the present application.

Where the target cell includes a nerve cell being treated, the vectorcan be delivered at an amount sufficient for the therapeutic peptide tobe expressed to a degree, which allows for highly effective therapy. Thetherapeutic peptide can be expressed for any suitable length of timewithin the target cell, including transient expression and stable,long-term expression. In one aspect of the application, the nucleic acidencoding the therapeutic peptide will be expressed in therapeuticamounts for a defined length of time effective to inhibitneurodegeneration in the subject being treated. In another aspect, thenucleic acid encoding the therapeutic peptide will be expressed intherapeutic amounts for a defined length of time effective to increasesurvival rate of neurons.

The therapeutic agents described herein may be modified (e.g.,chemically modified). Such modification may be designed to facilitatemanipulation or purification of the molecule, to increase solubility ofthe molecule, to facilitate administration, targeting to the desiredlocation, to increase or decrease half life. A number of suchmodifications are known in the art and can be applied by the skilledpractitioner.

In the methods of treatment disclosed herein, a therapeuticallyeffective amount of the therapeutic agent is administered to the subjectto treat a polyQ protein associated neurodegenerative disease. In oneembodiment, a formulation including the therapeutic agent can beadministered to the subject systemically in the period from the time of,for example, up to hours, days, and/or weeks after the disease ordisorder is diagnosed.

The therapeutic agents can be delivered to a subject by any suitableroute, including, for example, local and/or systemic administration.Systemic administration can include, for example, parenteraladministration, such as intramuscular, intravenous, intraarticular,intraarterial, intrathecal, subcutaneous, or intraperitonealadministration. The agent can also be administered orally,transdermally, topically, by inhalation (e.g., intrabronchial,intranasal, oral inhalation or intranasal drops) or rectally. In someembodiments, the therapeutic agent can be administered to the subjectvia intravenous administration using an infusion pump to deliver daily,weekly, or doses of the therapeutic agent.

Pharmaceutically acceptable formulations of the therapeutic agent can besuspended in aqueous vehicles and introduced through conventionalhypodermic needles or using infusion pumps.

For injection, therapeutic agent can be formulated in liquid solutions,typically in physiologically compatible buffers such as Hank's solutionor Ringer's solution. In addition, the therapeutic agent may beformulated in solid form and re-dissolved or suspended immediately priorto use. Lyophilized forms are also included. The injection can be, forexample, in the form of a bolus injection or continuous infusion (suchas using infusion pumps) of the therapeutic agent.

It will be appreciated that the amount, volume, concentration, and/ordosage of the therapeutic agent that is administered to any one animalor human depends on many factors, including the subject's size, bodysurface area, age, the particular composition to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. Specific variations of the above notedamounts, volumes, concentrations, and/or dosages of therapeutic agentcan be readily be determined by one skilled in the art using theexperimental methods described below.

In some embodiments, a therapeutic agent, such as a therapeutic peptidedescribed herein, can be administered can be administered locally and/orsystemically to a subject in need thereof at a dose or amount of about0.1 μmol, about 1 μmol, about 5 μmol, about 10 μmol, or more; or about0.0001 mg/kg, about 0.001 mg/kg, about 0.01 mg/kg, about 0.1 mg/kg, orabout 1 mg/kg to about 5 mg/kg or 10 mg/kg of the subject being treated.The therapeutic agent can be administered daily, weekly, biweekly,monthly or less frequently.

In some embodiments, the therapeutic agent can be administered to thesubject at an amount effective to increase plasma levels of NAD+, FAD,and/or citrate in the subject. As shown in the Examples, treatment withHV-3 in R6/2 mice significantly increased the plasma content of NAD⁺,FAD, and citrate (Table 1). NAD and FAD are central biomoleculesinvolved in energy production and mitochondrial metabolic activity;declines in NAD and FAD levels reflect decreased mitochondrial number,density, and activity. Thus, the findings here in parallel demonstratedthat mtHtt-induced mitochondria-accumulated VCP causes mitochondrialdysfunction and global energy deficits in HD, leading to neuronal celldeath. Because the depletion in NAD was noted in HD patient cells andblood, normalization of NAD content in HD mouse plasma by HV-3 treatmentcan provide a biomarker amenable for therapeutic intervention.

In another embodiment, the therapeutic agent can be administered to asubject systemically by intravenous injection or locally at the site ofinjury, usually within about 24 hours, about 48 hours, about 100 hours,or about 200 hours or more of when an injury occurs (e.g., within about6 hours, about 12 hours, or 24 hours, inclusive, of the time of theinjury).

In other embodiments, a pharmaceutically acceptable formulation used toadminister the therapeutic agent(s) can also be formulated to providesustained delivery of the active compound to a subject. For example, theformulation may deliver the active compound for at least one, two,three, or four weeks, inclusive, following initial administration to thesubject. For example, a subject to be treated in accordance with themethod described herein can be treated with the therapeutic agent for atleast 30 days (either by repeated administration or by use of asustained delivery system, or both).

Approaches for sustained delivery include use of a polymeric capsule, aminipump to deliver the formulation, a biodegradable implant, orimplanted transgenic autologous cells (see U.S. Pat. No. 6,214,622).Implantable infusion pump systems (e.g., INFUSAID pumps (Towanda, Pa.));see Zierski et al., 1988; Kanoff, 1994) and osmotic pumps (sold by AlzaCorporation) are available commercially and otherwise known in the art.Another mode of administration is via an implantable, externallyprogrammable infusion pump. Infusion pump systems and reservoir systemsare also described in, e.g., U.S. Pat. Nos. 5,368,562 and 4,731,058.

Vectors encoding the therapeutic peptides can often be administered lessfrequently than other types of therapeutics. For example, an effectiveamount of such a vector can range from about 0.01 mg/kg to about 5 or 10mg/kg, inclusive; administered daily, weekly, biweekly, monthly or lessfrequently.

The following examples are included to demonstrate different embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples, which follow representtechniques discovered by the inventor to function well in the practiceof the claimed embodiments, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the claims.

EXAMPLE 1

In this Example we show that VCP was aberrantly translocated to themitochondria where it was bound to mtHtt in a variety of HD models invitro and in vivo. Such accumulation of VCP on mitochondria resulted inexcessive mitophagy, mitochondrial mass loss, and subsequent neuronaldegeneration in HD models in culture and in animals. Significantly,blocking VCP translocation to mitochondria by a novel peptide HV-3 thatinterferes with VCP and mtHtt interaction inhibited VCP-mediatedmitophagy impairment, suppressed mitochondrial dysfunction, and reducedHD-associated neuropathology and motor deficits in two HD transgenicmouse models.

Materials and Methods

Antibodies and Reagents

Protein phosphatase inhibitor and protease inhibitor cocktails werepurchased from Sigma-Aldrich. VCP inhibitor Eer I and proteasomeinhibitor MG132 were from Tocris Bioscience. Antibodies for Tom20(sc-11415, 1:1000), c-Myc (sc-40, 1:1000), GFP (sc-9996, 1:1000), GST(sc-138, 1:500) and Parkin (sc-32282, 1:1000) were from Santa CruzBiotechnology. Full-length Htt (MAB2166, 1:1000), polyQ (MAB1574,1:1000), EM48 (MAB5374, 1:1000) and NeuN (MAB377, 1:500) antibodies werefrom Millipore. Pan-actin (A1978, 1:10,000) and Flag (F3165, 1:5000)antibodies were from Sigma-Aldrich. Antibodies for VDAC (ab14734,1:2000) and VCP (ab109240, 1:10000) were from Abcam. EEA1 (3288, 1:500)and LC3 (2775, 1:1000) antibodies were from Cell Signaling, WFS1(NB100-1918, 1:1000) antibody was from Novus, HMGB1 (high-mobility groupbox B1, 10829-1-AP, 1:1000) antibody was from Proteintech, and GRP78(ADI-SPA-826, 1:1000) and Calnexin (ADI-SPA-860, 1:1000) antibody wasfrom Enzo Life Sciences. Anti-mouse IgG and anti-rabbit IgG,peroxidase-linked, species-specific antibodies were fromThermo-Scientific. The Htt/VCP peptides were synthesized by AmericanPeptide Company, Inc., and conjugated to TAT-carrier peptide (aminoacids 47-57) for transmembrane delivery. Note that TAT₄₇₋₅₇-baseddelivery was used in culture and in vivo and was found to be safe andefficacious for delivery of peptide cargoes to cells and also to crossthe blood-brain barrier.

Constructs and Transfection

Myc-tagged full-length Htt with 23Q or 73Q plasmid was obtained from theCHDI foundation. The full-length VCP wild-type (VCP^(WT)) and GFP-LC3Bplasmids were obtained from Addgene. To construct themitochondria-targeting VCP plasmid, CMV-mito-GEM-GECO1 was digested withBamHI and Hind III, and VCP was PCR-amplified and inserted into theplasmid backbone. Site mutation of the VCP plasmid was performed using asite-mutagenesis kit (Agilent Technologies, Inc.). Cells weretransfected with TransIT®-2020 (Mirus Bio LLC) following themanufacturer's protocol.

Cell Culture

Immortalized striatal cell lines HdhQ111 mutant and HdhQ7 wild-typederived from striatal cells from HdhQ111/111 and HdhQ7/Q7 knock-intransgenic mice (expressing 111 and 7 glutamine repeats, respectively)were obtained from the CHDI Foundation. Cells were cultured in DMEMsupplemented with 10% FBS, 100 μg/ml penicillin, 100 μg/ml streptomycin,and 400 μg/ml G418. Cells were grown at 33° C. in a 5% CO₂ incubator.Cells within 14 passages were used in all experiments.

Human cervix carcinoma cells (HeLa cells) and HEK293 cells weremaintained in DMEM supplemented with 10% FBS and 1% (v/v)penicillin/streptomycin.

Primary striatal neurons from E18 rat midbrain tissue (BrainBits,Springfield, Ill., USA) were seeded on cover slides that were coatedwith poly-D-lysine/laminine and grown in neurobasal medium supplementedwith 2% B27 and 0.5 mM glutamate. At 7 DIV, cells were transfected withthe control vector or flag-VCP^(mt) using TransIT®-2020 TransfectionReagent combined with formulated BrainBits transfection media forprimary neurons (BrainBits, USA).

iPS cells from normal subjects and HD patients were differentiated intoneurons using the protocol from our previous studies. Neurons (about5,000 cells) were plated onto 12-mm poly-D-lysine/laminine-coatedcoverslips and grown in 24-well plates in neuronal differentiationmedium as described previously.

All of the above cells were maintained at 37° C. in 5% CO₂-95% air.

RNA Interference

For silencing Htt and VCP in HD striatal cells, control siRNA, mouse Httand mouse VCP siRNA were purchased from Thermo Fisher Scientific. HdhQ7and HdhQ111 cells were transfected either with control siRNA, or Htt orVCP siRNA using TransIT-TKO® Transfection Reagent (Mirus Bio LLC),according to the manufacturer's instructions.

Isolation of Mitochondria-Enriched, ER-Enriched and Cytosolic Fractions

Cells were washed with cold PBS and incubated on ice for 30 minutes in alysis buffer (250 mM sucrose, 20 mM HEPES-NaOH, pH 7.5, 10 mM KCl, 1.5mM MgCl₂, 1 mM EDTA, protease inhibitor cocktail and phosphataseinhibitor cocktail). Mice brains were minced and homogenized in lysisbuffer and then placed on ice for 30 minutes. Collected cells or tissuewere disrupted 20 times by repeated aspiration through a 25-gaugeneedle, followed by a 30-gauge needle. The homogenates were spun at 800g for 10 minutes at 4° C., and the resulting supernatants were spun at10,000 g for 20 minutes at 4° C. The pellets were washed with lysisbuffer and spun at 10,000 g again for 20 minutes at 4° C. The finalpellets were suspended in lysis buffer containing 1% Triton X-100 andwere mitochondrial-rich lysate fractions. The supernatant wascentrifuged at 100,000 g, 4° C., for 1 hour, the pellets were suspendedin lysis buffer containing 1% Triton X-100 as ER fractions. The finalsupernatant was cytosolic fractions. The mitochondrial proteins VDAC andthe ER protein WFS1 were used as loading controls for mitochondria andER fractions, respectively.

Immunoprecipitation

Cells were lysed in a total cell lysate buffer (50 mM Tris-HCl, pH 7.5,containing 150 mM NaCl, 1% Triton X-100, and protease inhibitor) or in amitochondrial isolation buffer (250 mM sucrose, 20 mM HEPES-NaOH, pH7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, protease inhibitor cocktail,and phosphatase inhibitor cocktail). Total lysates or mitochondriallysates or the mixture of ER and cytosolic fractions were incubated withthe indicated antibodies overnight at 4° C. followed by the addition ofprotein A/G beads for 1 hour Immunoprecipitates were washed four timeswith cell lysate buffer and were analyzed by SDS-PAGE andimmunoblotting.

Rational Design of Peptide Inhibitor

Two nonrelated proteins that interact in an inducible manner have oftenshared short sequences of homology that represent sites of both inter-and intra-molecular interactions. Similar to the peptide design for PKCpeptide deltaV1-1 and Drp1 peptide P110, we used L-ALIGN sequencealignment software and identified two different regions of homologybetween VCP (VCP, Human, AAI21795) and Htt (Htt, human, NP_002102).These regions are marked as regions HV from 1 to 4. We found that allthe homologous sequences are conserved in a variety of species includinghuman, mouse, rat and fish. We synthesized the four peptidescommercially corresponding to regions HV1-4 and conjugated them to thecell permeating TAT protein-derived peptide, TAT₄₇₋₅₇, as we described.These peptides are referred to as HV-1, HV-2, HV-3 and HV-4. The puritywas assessed as >98% by mass spectrometry. Lyophilized peptides weredissolved in sterile water and stored at −80° C. until use.

Measurement of Cell Viability

HdhQ7 and Q111 mouse striatal cells were treated with the HV-3 peptideor the control peptide TAT (3 μM, each) in an FBS-free DMEM medium or inDMEM containing 10% serum for 24 hours. Medium from the cultured cellswas harvested. Proteins from the medium were purified using Amicon Ultra0.5 ml centrifugal filters (Millipore). HMGB1 release into the mediumwas then analyzed by Western blotting with anti-HMGB1 antibody. Inparallel, cell death was determined by measuring LDH release into theculture medium, as described previously.

Immunocytochemistry

Cells cultured on coverslips were washed with cold PBS and fixed in 4%formaldehyde, and then permeabilized with 0.1% Triton X-100. Afterincubation with 2% normal goat serum (to block nonspecific staining),fixed cells were incubated overnight at 4° C. with indicated primaryantibodies. Cells were washed with PBS and incubated for 60 minutes withAlexa Fluor 568, 488 or 405 secondary antibody, followed by incubationwith Hoechst dye (1:10,000; Invitrogen) for 10 minutes. Coverslips weremounted, and slides were imaged by confocal microscopy (Fluoview FV100;Olympus).

To determine mitochondrial mass in cultures, cells were stained withantibodies against Tom20 or stained with Mitotracker green. Thefluorescent density of Tom20 (1:500) or mitotracker green wasquantitated using NIH Image J software. To measure the membranepotential of mitochondria in cultures, cells were incubated with 0.25 μMtetra-methyl rhodamine (TMRM) (Invitrogen Life Science) for 20 minutesat 37° C. To determine lysosomal activity, cells were incubated withLyso-ID Red dye (Enzo Life Science) for 30 min at 37° C. The images werevisualized by microscope and quantitation of the density of redfluorescence was carried out using NIH ImageJ software as describedpreviously. For immunocytochemistry study, at least 100 cells/group werecounted and quantitated by an observer blind to experimental conditions.

In patient-iPS cell-derived neurons, to ensure the observation ofmitochondria in the medium spiny neurons, the cells were stained with amitochondrial marker (anti-TOM20, 1:500) and markers for medium spinyneurons (DARPP-32, 1:200, Epitomics), as we previously described. Atleast 50 neurons/group were counted and quantitated.

Animal Model of HD

All experiments in animals were conducted in accordance with protocolsapproved by the Institutional Animal Care and Use Committee of CaseWestern Reserve University and were performed based on the NationalInstitutes of Health Guide for the Care and Use of Laboratory Animals.Sufficient procedures were employed for reducing pain or discomfort ofsubjects during the experiments.

Male R6/2 mice and their wild-type (WT) littermates were purchased fromJackson Laboratories [Bar Harbor, Me.; B6CBA-TgN (HD exon1)62; JAX stocknumber: 006494]. These mice are transgenic for the 5′ end of the humanHD gene carrying 100-150 glutamine (CAG) repeats.

YAC128 [FVB-Tg(YAC128)53Hay/J, JAX stock number: 004938] breeders werepurchased from Jackson Laboratories. The YAC128 mice contain afull-length human huntingtin gene modified with a 128 CAG repeatexpansion in exon 1. The mice were mated, bred, and genotyped in theanimal facility of Case Western Reserve University. Male mice were usedin the study.

All of the mice were maintained with a 12-hour light/dark cycle (on 6am, off 6 pm).

Systemic Peptide Treatment in HD Mice

All randomization and peptide treatments were prepared by anexperimenter not associated with behavioral and neuropathology analysis.

Male hemizygous R6/2 mice (Tg) and their age-matched wild-typelittermates (Wt) were implanted with a 28-day osmotic pump (Alzet,Cupertino Calif.) containing TAT control peptide or HV-3 peptide, whichdelivered peptides to the mice at a rate of 3 mg/kg/day. The first pumpwas implanted subcutaneously in the back of 5-week-old mice between theshoulders and replaced once, after 4 weeks.

YAC128 mice (Tg) and their age-matched wild-type littermates (WT) wereimplanted with an osmotic pump containing TAT control peptide or HV-3peptide (3 mg/kg/day, each) starting from the age of 3 months. The pumpwas replaced once every month. By the age of 12 months, the treatmentswere terminated and the mouse samples were harvested for analysis.

Behavioral Analysis in HD Mice

All behavioral analyses were conducted by an experimenter who was blindto genotypes and treatment groups.

Activity Chamber

Gross locomotor activity was assessed in R6/2 mice and age-matchedwild-type littermates at the ages of 13 weeks and in YAC128 mice andage-matched wild-type littermates at the ages of 2, 3, 6, 9, and 12months. In an activity chamber (Omnitech Electronics, Inc), mice wereplaced in the center of the chamber and allowed to explore while beingtracked by an automated beam system (Vertax, Omnitech Electronics Inc).Distance moved, horizontal, vertical, and rearing activities wererecorded. Because R6/2 mice were sensitive to changes in environment andhandling, we only conducted one-hour locomotor activity analysis forR6/2 mice and wild-type littermates. We performed 24 hours of locomotoractivity analysis for YAC128 mice and their wild-type littermates.

Clasping Behavior

Hindlimb clasping was assessed with the tail suspension test once a weekfrom the ages 8 to 11 weeks in R6/2 mice. Mice were suspended by thetail for 60 seconds and the latency for the hindlimbs or all four pawsto clasp was recorded using the score system: Clasping over 10 seconds,score 3; 5-10 seconds, score 2; 0-5 seconds, score 1; 0 seconds, score0.

Rotarod Analysis

The motor coordination and balance of YAC128 mice were tested on anaccelerating Rotarod (IITC Life Sciences, Serials 8) at the ages of 2,3, 6, 9 and 12 months. Training and baseline testing for motor functiontasks were conducted at 2 months of age. For training, mice were giventhree 120-second trials per day at a fixed-speed of 15 rpm for threeconsecutive days. During the testing phase, the Rotarod accelerated from5 to 40 rpm over 3 minutes; the maximum score was 300 seconds. Rotarodscores were the average of three trials per day (with 2 hours restbetween trials) for 3 consecutive days.

The body weight and survival rate of HD mice and wild-type littermateswere recorded throughout the study period.

Immunohistochemistry and Stereological Measurements

Mice were deeply anesthetized and transcardially perfused with 4%paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). Brains wereprocessed for paraffin embedment. Brain sections (5 μm, coronal) wereused for immunohistochemical localization of DARPP-32 (1:500, Epitomics)using the IHC Select HRP/DAB kit (Millipore). Quantitation of DARPP-32immunostaining was described in our previous study. The same imageexposure times and threshold settings were used for sections from alltreatment groups.

To measure the number of NeuN-positive cells, a series of 25 mm thickcoronal sections spaced 200 mm apart spanning the striatum were stainedwith NeuN antibody (Millipore, 1:500) and visualized bydiaminobenzidine. For neuropathological analyses, brain sections wereanalyzed stereologically as described previously. Briefly, unbiasedstereological counts of NeuN-positive neurons within the striatum wereperformed using unbiased stereological principles and analyzed withStereoInvestigator software (Microbrightfield, Williston, Vt.). Opticalfractionator sampling was carried out on a Leica DM5000B microscope(Leica Microsystems, Bannockburn, Ill.) equipped with a motorized stageand Lucivid attachment (40× objective). The following parameters wereused in the final study: grid size, (X) 500 μm, (Y) 500 μm; Countingframe, (X) 68.2 μm, (Y) 75 μm, depth was 20 μm. Gundersen coefficientsof error for m=1 were all less than 0.10. Stereologic estimations wereperformed with the same parameters in striatum of wt or YAC transgenicmice treated with the control peptide or peptide HV-3 (n=6 mice/group).The total volume of stratial tissue measured in each brain is calculatedby StereoInvestigator and the neuronal density is presented as Neu-Npositive cell number per mm³.

Quantitation was conducted by an experimenter blind to the experimentalgroups.

Electron Microscopy

Small pieces of the striata tissue were fixed by immersion in triplealdehyde-DMSO. After rinsing in 0.1 M phosphate buffer (pH 7.3), thetissues were post-fixed in ferrocyanide-reduced osmium tetroxide. Waterrinse was followed by overnight soaking in acidified uranyl acetate.After rinsing in distilled water, the tissue blocks were dehydrated inascending concentrations of ethanol, passed through propylene oxide, andembedded in Poly/Bed resin. Thin sections were sequentially stained withacidified uranyl acetate followed by a modification of Sato's triplelead stain. These sections were examined in a FEI Tecnai Spirit (T12)transmission electron microscope with a Gatan US4000 4 k×4 k CCD at theCase Western Reserve University EM core facility. Mitochondria from 15random areas in each animal were imaged by an experimenter blind to theexperimental groups. The number of mitophagosomes per 100 um² wascounted.

Western Blot Analysis

Protein concentrations were determined by Bradford assay. Protein wasthen resuspended in Laemmli buffer, loaded on SDS-PAGE, and transferredonto nitrocellulose membranes. Membranes were probed with the indicatedantibody, followed by visualization with ECL.

Statistical Analysis

Sample sizes are determined by power analysis based on pilot datacollected by our labs or published studies. In animal studies, we usedn=15-18 mice/group for behavioral tests, n=6 mice/group for biochemicalanalysis and n=6 mice/group for pathology studies. In cell culturestudies, we performed each study with three independent replications.For all of the animal studies, we have ensured randomization and blindedconduct of experiments. For all imaging analysis, the quantitation wasconducted by an observer who was blind to the experimental groups.

Data were analyzed by Student's t test or one-way ANOVA with post-hocTukey test or One-way ANOVA with Scheffe post hoc test for comparisonbetween two groups. Survival and body weight were analyzed byrepeated-measures two-way ANOVA. Data are expressed as mean±SEM.Statistical significance was considered achieved when the value of p was<0.05.

Results

VCP was Recruited to Mitochondria by mtHtt in HD

We used HD mouse striatal HdhQ111 (mutant) and HdhQ7 (wild-type) cellsto profile the interactome of mtHtt on mitochondria (FIG. 1A). HdhQ111and Q7 cells were immortalized from knock-in mice carrying 111 and 7CAG, respectively, in the mouse htt gene. These cell lines have beenwidely used as a cell culture model to study HD. We isolatedmitochondria from these cultured striatal cells, and conductedimmunoprecipitation (IP) of mitochondrial fractions with anti-MAB2166antibody that recognizes both wild-type (wt) and mutant (mt) Htt. Tandemmass spectrometry analysis following affinity purification identified 9proteins that putatively bound to mitochondria-associated mtHtt inHdhQ111 but not wt Htt in HdhQ7 striatal cells (FIG. 1A). Among theseproteins, VCP was the leading candidate that bound to mtHtt onmitochondria of HdhQ111 cells (FIG. 1A).

Before validating this proteomic analysis on the interaction between VCPand mtHtt, we first want to confirm whether VCP is localized onmitochondria in models of HD. Western blot analysis of cellularfractionations revealed that VCP was markedly enriched in themitochondria of HdhQ111 cells relative to those in HdhQ7 cells (FIG.1B), while there was no increase in the recruitment of VCP to the ER inHdhQ111 cells when compared to that of HdhQ7 cells (FIG. 1B). Reductionof mtHtt levels by treatment of HdhQ111 cells with Htt small interferingRNA (siRNA) abolished VCP translocation to the mitochondria (FIG. 1C),indicating that mtHtt is required for VCP recruitment to themitochondria. Confocal imaging analysis consistently showed increasedlocalization of VCP on mitochondria, but not on the ER and endosome ofHdhQ111 striatal cells, relative to that in HdhQ7 cells (FIG. 1D). Asimilar enrichment of VCP on mitochondria was further observed inmitochondrial fractions isolated from the striatum of both R6/2 mice atthe age of 9 weeks and YAC128 mice at the age of 6 months (FIG. 1E). Totest whether VCP accumulation on mitochondria also exists in human HD,we analyzed VCP localization on mitochondria by confocal microscopy inthe caudate nucleus of postmortem brains from three HD patients andthree normal subjects. We observed greater localization of VCP onmitochondria in HD patient brains than in normal subjects (FIG. 1F).These data collectively demonstrate that VCP is recruited to andaccumulated on mitochondria in HD. Because there was no evidence ofincreased VCP to mitochondria in response to Parkinson'sdisease-associated mutants, VCP recruitment to mitochondria is likely tobe disease- or stress-dependent.

Next, we isolated mitochondria, ER, and cytosolic fractions from HdhQ7and HdhQ111 mouse striatal cells, and conducted IP with anti-VCPantibody followed by immunoblotting (IB) with anti-MAB2166 antibody. Weobserved mtHtt proteins in VCP immunoprecipitates of mitochondrialfractions but not in that of ER and cytosolic fractions in HdhQ111 mousestriatal cells (FIG. 1G-left panel). Although VCP interacted with wt Htton the mitochondria in HdhQ7 striatal cells, the extent is much lesserthan that in HdhQ111 cells only expressing mtHtt (FIG. 1G-left panel).To further validate the interaction between VCP and mtHtt, we performedIP analysis with anti-VCP antibody followed by IB with anti-1C2 antibodythat recognizes only expanded polyQ proteins. As shown in FIG. 1G-rightpanel, VCP only bound to mtHtt in mitochondrial fractions, not in ER orcytosolic fractions of HdhQ111 striatal cells, even though mtHtt wasexpressed in the ER and cytosolic fractions. Consistently, mtHttrecognized either by anti-1C2 antibody or by anti-EM48 antibody [Note:EM48 antibody preferentially reacts with full-length human mtHtt in mice(40)] was observed in VCP immunoprecipitates of mitochondrial fractionsisolated from the striata of YAC128 mice at the age of 6 months (FIG.1H). Again, there was no obvious binding of VCP and mtHtt observed inthe ER or cytosolic fractions of YAC128 mouse striata (FIG. 1H). Arecent proteomic analysis of the Htt interactome in total brain lysatesof BACHD transgenic mice supported our finding that VCP is a bindingprotein of Htt and that increased interaction between VCP and mtHtt isrelevant to HD. Now we are able to locate this interaction withmitochondria in models of HD in culture and in animals.

VCP plays a central role in protein degradation by binding to itssubstrates. We found that treatment with either MG132, a proteasomeinhibitor to prevent protein degradation, or Eeyarestatin I (Eer I), aninhibitor that blocks VCP substrate degradation, did not affect Htt ormtHtt protein levels in HdhQ or HdhQ111 cells, respectively. These dataexcludes the possibility that Htt or mtHtt is a substrate of VCP onmitochondria.

Together, our findings show that VCP is selectively recruited tomitochondria in models of HD where it interacts with mitochondria-boundmtHtt.

Development of a Peptide Inhibitor to Interfere with Htt/VCP Interaction

We used L-ALIGN sequence alignment software to identify two differentregions of homology between VCP (human, AAI21795) and Htt (human,NP_002102) (FIG. 2A). The four regions are marked as regions HV-1 toHV-4 (FIG. 2B). We synthesized peptides corresponding to the fourhomologous regions between VCP and Htt (FIG. 2A), and conjugated them tothe cell permeating TAT protein-derived peptide, TAT47_57, to enable invivo delivery, as we previously described. These peptides are referredto as HV-1, HV-2, HV-3, and HV-4. By incubating these peptides with amixture of GST-VCP and total lysates of mouse brains (expressingfull-length Htt) followed by GST pull down analysis, we found that onlythe addition of peptide HV-3 blocks the interaction of VCP/Htt in thisin vitro binding assay (FIG. 8A). In HEK293 cells co-expressingMyc-tagged full-length Htt with 23 or 73 CAG repeats (Myc-23Q FL orMyc-73Q FL, respectively) and GFP-VCP, consistent with our observationshown in FIG. 1G and HD, VCP was preferentially bound to Myc-73Q FL(mtHtt) over Myc-23Q FL (FIG. 2C-left panel). Of four peptides, onlytreatment with HV-3 peptide (3 μM/day for two days) greatly blocked theVCP/mtHtt interaction in Myc-73Q FL expressing cells (FIG. 2C, FIG. 8B).

Peptide HV-3 is derived from the Htt c-terminal and corresponds to thesequence in the D1 domain of VCP (FIGS. 2A and B). The sequence of HV-3in Htt is highly conserved among species (FIG. 8C). In addition to Httand VCP, there is no sequence identity or similarity found between HV-3and other proteins by the BLAST (basic local alignment search tool)analysis. Molecular docking analysis indicated that the peptide HV-3 isbound to the surface of VCP structure (FIG. 8D). Deletion of thesequence in VCP corresponding to HV-3 peptide abolished the interactionbetween Htt and VCP (FIG. 8E). These data suggest that HV-3 mayrepresent an important interaction region for VCP in Htt. Therefore, weselected HV-3 as a peptide candidate that interferes with VCP/mtHttbinding.

In HdhQ7 and HdhQ111 mouse striatal cells, we treated cells with HV-3 orcontrol peptide TAT (3 μM/day for 3 days, each) and determined VCPmitochondrial levels. Treatment with HV-3 abolished VCP translocation tomitochondria in HdhQ111 cells relative to cells treated with TAT, whileHV-3 had minor effects on VCP mitochondrial levels in HdhQ7 cells (FIG.2D). In YAC128 mice which express a full-length human mtHtt, we treatedthe mice with peptide HV-3 or control peptide TAT (3 mg/kg/day, each)using an osmotic mini pump starting at the age of 3 months.Consistently, the treatment abolished VCP translocation to themitochondria of the striatum in YAC 128 mice at the age of 6 monthsrelative to YAC128 mice treated with control peptide TAT (FIG. 2E). Notethat a FITC-positive signal was observed in neurons of mouse brain after2 days of continual systemic delivery of FITC-conjugated HV-3,supporting our idea that the peptide HV-3 can enter and accumulate incells of brains. Unlike YAC128 mice, HD R6/2 mice express exon 1 of thehuman HD gene carrying more than 120 CAG repeats. Surprisingly, HV-3treatment still blocked the VCP translocation to the mitochondria thatoccurred in the R6/2 mouse striatum (FIG. 2F). The toxic form of Htt(N-terminal fragment Htt) undergoes conformational rearrangement thatleads to intramolecular proximity between the N domain and thepolyproline region of Htt at the C-terminal. It is possible that such aconformational change results in the binding of VCP to both thefull-length and the fragment of Htt. HV-3 may block the VCP accumulationon the mitochondria by disrupting the interaction between VCP and thecomplex of fragment/full-length Htt. Finally, we found that HV-3treatment had no effects on VCP total protein levels in cultured HDmouse striatal cells or in the brains of R6/2 and YAC128 mice.

Suppression of VCP Association with Mitochondria by Peptide HV-3 ReducedMitochondrial Damage and Cell Death in HD Mouse- and Patient-DerivedCells

We next examined the effects of HV-3 treatment on mitochondrial functionand cell survival in HD cell cultures. Mitochondrial depolarization andmitochondrial fragmentation are featured in experimental models of HDand human HD. As shown in FIGS. 3A and B, treatment with HV-3 markedlyimproved the mitochondrial membrane potential (MMP) and reduced thenumber of fragmented mitochondria in HdhQ111 striatal cells, compared tothe cells treated with control peptide TAT, suggesting a reduction ofmitochondrial damage. In HdhQ111 striatal cells subjected to 24 hours ofserum withdrawal, HV-3 treatment reduced the release of high mobilitygroup box 1 (HMGB1) and lactate dehydrogenase (LDH), two indicators ofcell death (FIGS. 3C and D). However, HV-3 had no effects on apoptosis,as evaluated by the activity of caspase-3, under the same culturedconditions. VCP is an essential regulator of the ERAD and ER-relatedunfolded protein response. We found that HV-3 treatment had no effectson the ER stress response, excluding the possibility that the protectionprovided by HV-3 on mitochondria is the result of a secondaryconsequence of the inhibition of ER stress.

Neurons derived from HD patient induced pluoripotent stem cells (HD-iPScells) exhibited mitochondrial damage and increased cell death. Inneurons immuno-positive for both anti-DARPP-32 (a marker of medium spinyneurons) and anti-Tubulin β-III (a marker of mature neurons), treatmentwith HV-3 (1 μM/day for 5 days starting 30 days after initiation ofneuronal differentiation) significantly reduced neurite shorteningcompared to patient neurons treated with control peptide TAT (FIG. 4E,F). Further, HV-3 treatment suppressed neuronal cell death in thesepatient-derived neurons subjected to growth factor withdrawal (FIG. 4H).The neuroprotective effects of HV-3 were consistently associated withimproved mitochondrial membrane potential and mitochondrial length alongneurite (FIG. 3G). Taken together, the above results demonstrate thattreatment with HV-3 protects against mitochondrial damage and neuronalcell death under HD-associated conditions.

Interestingly, we found that the peptide HV-3 had only minor effects onVCP mitochondrial levels, mitochondrial membrane potential, andmorphology, as well as cell survival rate in wild-type counterparts ofthe above HD experimental models (FIGS. 2, 3), which is likely theresult of less binding between VCP and wt Htt under basal conditions(FIG. 1G). Normal and mutant polyglutamine proteins interact with VCPand only mutant proteins specifically affect dynamism of VCP and impairits function, thus it is also possible that disruption of wtHtt/VCPinteraction results in minor physiological impact.

Collectively, our findings not only show that mtHtt is required for VCPrecruitment to mitochondria, but also suggest that HV-3 is useful fortesting the biological significance of mitochondria-accumulated VCP inHD.

VCP Translocation to Mitochondria by mtHtt Impaired Mitochondria-RelatedAutophagy in HD Models In Vitro and In Vivo

How does mtHtt-induced VCP mitochondrial accumulation mediatemitochondrial dysfunction and cell death? Apoptosis and autophagic celldeath are manifested in HD neuropathology. Blocking VCP recruitment tomitochondria by treatment with HV-3 did not affect apoptotic cell death.In contrast, down-regulation of VCP by VCP siRNA in mouse HdhQ111striatal cells reduced the levels of mitochondria-associated LC3 II, amarker of mitophagy, (FIG. 4A). Moreover, expression of Flag-VCP inwild-type mouse striatal cells induced GFP-LC3B association withmitochondria, which could be inhibited by treatment with HV-3 (FIG. 4B).Thus, we speculated that mtHtt-induced VCP accumulation on mitochondriatriggers mitochondria-associated autophagy.

We treated HdhQ7 and HdhQ111 striatal cells with peptide HV-3 andcontrol peptide TAT, and determined autophagic activity in these cells.In HdhQ111 cells, we observed an increased number of GFP-LC3B puncta, aspecific marker for autophagosomes, and hyperactivity of lysosome enzymeCathepsin B, both of which were reduced by treatment with HV-3 (FIG. 4C,D). Similarly, neurons derived from HD patient-iPS cells exhibited lowermitochondrial mass and lysosome hyperactivity, whereas treatment withHV-3 corrected these aberrant events (FIG. 4E, F). Further, we examinedthe ultrastructure of striatal mitochondria in YAC128 mice. Relative tothat in wild-type littermates, we observed an increase in the number ofmitophagosomes in 9-month-old YAC128 mice treated with the controlpeptide TAT, which was reduced by HV-3 treatment (FIG. 4G). The abovefindings suggest that inhibition of VCP mitochondrial accumulation in HDby treatment with peptide HV-3 suppress excessive mitophagy and improvemitochondrial quality.

LC3 in mammals or Atg8 in yeast play a key role in both autophagosomemembrane biogenesis and cargo recognition. In yeast, Atg32 functions asa receptor on mitochondria to initiate mitophagy through interactionwith Atg8. Similarly, mammalian mitophagic adaptors, such as FUNDC1,p62, BNIP3, and AMBRA1, all bind to LC3 via a typical linear motif witha core consensus sequence of W/Y/F xx L/I/V, also called LC3-interactingregion (LIR). Given the above findings, we hypothesized that VCP mightbind to LC3 on mitochondria to enhance mitophagosome production. Usingan iLIR server, we found that VCP contains two segments of sequence(LEAYRPIR (SEQ ID NO: 11) and AVEFKVVE (SEQ ID NO: 12)) located in the13 stands of the N terminal (FIG. 5A) that fulfill the characteristicsof the LIR. To determine if VCP binds to LC3 via putative LIR motifs, wegenerated two mutants (VCP-YI^(AA) and VCP-FV^(AA)) in which Y/I and F/Vwere all replaced by alanine. Because of higher transfection efficiency,we first used HeLa cells to determine the effects of these mutants. InHeLa cells co-expressing Myc-VCP and GFP-LC3B, we performed IP analysisof mitochondrial fractions with anti-GFP antibody followed by IB withanti-Myc antibody. We found that Myc-VCP was bound to GFP-LC3B in themitochondrial fractions of cells (FIG. 5B). Expression of eitherVCP-YI^(AA) or VCP-FV^(AA) abolished the VCP/LC3 interaction relative tocells expressing VCP^(WT) (FIG. 5B). Moreover, expression of VCP-YI^(AA)or VCP-FV^(AA) reduced LC3 association with the mitochondria (FIG. 5C)and increased mitochondrial mass (FIG. 5D) compared to cells expressingVCP^(WT). These data demonstrate that mitochondria-accumulated VCPaccelerates mitophagy by interacting with LC3 through the LIRs.

To determine direct consequence of VCP mitochondrial accumulation onmitophagy and cell survival, we generated a construct encoding VCP fusedto a flag-vector containing a mitochondrial targeting sequence (MTS)(flag-VCP^(mt)). In HeLa cells expressing flag-VCP^(mt), we confirmedthe enrichment of flag-VCP^(mt) on mitochondria. We further observedthat expression of flag-VCP^(mt) induced a relocalization of themitochondria network, forming mitochondrial aggregates which is anintermediate step of mitophagy, around the perinuclear envelope. Theoccurrence of mitochondrial aggregates in cells expressing flag-VCP^(mt)increased by approximately sevenfold relative to cells not expressingflag-VCP^(mt). Moreover, the presence of flag-VCP^(mt) in cellsdecreased mitochondrial membrane potential and mitochondrial mass, butinduced an increase in the percentage of GFP-LC3B colocalizing withTom20-labeled mitochondria. Upon treatment with bafilomycin A (BFA) toprevent autophagosome-lysosome fusion, flag-VCP^(mt) expression elevatedthe autophagic flux of the mitochondria, indicating increased rate ofmitochondrial degradation. We then expressed the flag-VCP^(mt) andwild-type vector in rat primary striatal neurons. Expression offlag-VCP^(mt) elicited mitochondrial aggregates and caused neuriteshortening of medium spiny neurons that were labeled by anti-DARPP-32antibody (FIG. 5E, F). Thus, mitochondria-accumulated VCP causedmitochondrial and neuronal damage, at least in part, via impairment ofthe mitophagic process.

Treatment with HV-3 Reduced Behavioral Phenotypes of HD in HD R6/2 andYAC128 Mice

We next examined if blocking VCP accumulation on the mitochondriaprovides neuroprotection in in vivo animal models of HD. We treated HDR6/2 mice and YAC128 mice with control peptide TAT or peptide HV-3 usingthe protocols shown in Supplemental FIG. 5.

We first examined the effects of HV-3 treatment on behavioral phenotypesof HD in both R6/2 and YAC128 mice. HD R6/2 mice treated with thecontrol peptide TAT exhibited decreased horizontal and verticalactivities as well as less total traveled distance in the test ofspontaneous locomotion when evaluated at the age of 13 weeks, whereastreatment with HV-3 dramatically corrected these motor deficits (FIG.6A). The severity of clasping behavior in R6/2 mice treated with HV-3was significantly lower than those treated with the control peptide TATover the four-week observation period (FIG. 6A). HV-3 treatment alsoresulted in increased body weight and survival rate of R6/2 mice (FIG.6B, C). The treatment had no effects on motor ability, body weight, orlife span in wild-type mice (FIG. 6A-C), suggesting a lack of toxicityof the peptide treatment.

The YAC128 mice exhibit progressive motor abnormalities as well aslate-stage selective striatal neuron loss, closely recapitulating achronic feature of neuropathology seen in human HD. We examined thelong-term treatment effects of HV-3 on the behavioral outcomes in YAC128mice. Consistent with previous studies, YAC128 mice progressivelyexhibited deficits in motor activities; they showed gradually decreasingmotor coordination activity on the rotarod and defects in generalmotility examined by locomotor activity chambers. Significantly,sustained treatment with HV-3 improved general movement activity androtarod performance of YAC128 mice starting at the age of 6 months, andthe protection was lasted until the age of 12 months (FIG. 6D, E).Again, the treatment did not affect motor activity in wild-type micefrom 3 to 12 months of age.

Sustained Treatment with HV-3 Reduced Neuropathology in HD R6/2 andYAC128 Mice

In HD patients, medium spiny neurons in the striatum are particularlysusceptible to degeneration. The levels of dopamine signaling protein,DARPP-32, enriched in these cells are decreased in the striatum of HDpatients and mouse models. Thus, DARPP-32 has been used as a marker toassess the neuronal degeneration in HD mouse models. Western blotanalysis of striatal extracts revealed a significant reduction ofDARPP-32 protein levels in both R6/2 and YAC128 mice. HV-3 treatmentsignificantly increased DARPP-32 levels in the two mouse models (FIG.7A). In HD R6/2 mice, we consistently observed a decrease in the areaoccupied by DARPP-32-immunostained cells in the striatum, which wasincreased by HV-3 treatment (FIG. 7B, C). To further assess whether HV-3treatment can suppress neurodegenerative pathology in HD, we conductedunbiased stereology analyses to measure the number of striatal neuronsin YAC128 mice at the age of 12 months. We found that treatment withHV-3 significantly increased the number of neurons positive foranti-NeuN immunostaining in the dorsolateral striatum (FIG. 7D).

The findings from both the fragment and full-length mtHtt transgenicmouse models demonstrate that blocking mtHtt-induced VCP mitochondrialaccumulation by peptide HV-3 greatly reduced neuropathology and motordeficits that are associated with HD.

HV-3 Dose Response in Animal Models of R6/2 Mice

R6/2 mice express a fragment of mutant Huntingtin and the mouse lineexhibits severe neuropathology of Huntington's disease. The R6/2 micehave short life span and most of animals die before 15 weeks of age. Wedetermined the survival rate of R6/2 mice by the age of 13 weeks. Wetreated the R6/2 mice with HV-3 at 0.5, 1 and 3 mg/kg/day using osmoticmini pump (subcutaneously administration), and found that HV-3 at 3mg/kg/day dramatically improved the survival of R6/2 mice (FIG. 9).Therefore, we used 3 mg/kg/day of HV-3 in our rest of studies. Towildtype mice, we treated mice with either TAT or HV-3 at 3 mg/kg/day.There was no difference on survival rate in the groups treated with TATor HV-3.

EXAMPLE 2

Treatment with HV-3 Corrected Aberrant Mitochondrial Intermediates in HDMouse Plasma

Deficits in energy metabolism, attributable to mitochondrialdysfunction, is an early event in HD patients. To determine the effectsof HV-3 treatment on mitochondrial metabolic activity in vivo, we usedtarget metabolomics analysis of HD mouse plasma to profile theintermediates of the mitochondrial tricarboxylic acid (TCA) cycle, acentral metabolic pathway within mitochondria for eukaryotes. LC-MS/MSanalysis revealed that 8 out of 16 intermediates derived from the TCAcycle were decreased in R6/2 mouse plasma relative to those in wild-typemice (Table 1), suggesting a decrease in mitochondrial metabolicactivity. Treatment with HV-3 in R6/2 mice significantly increased theplasma content of NAD⁺, FAD, and citrate (Table 1). In contrast, HV-3treatment had no effects on the intermediates derived from amino acidmetabolism, although some of intermediates in this pathway were alteredin HD mice relative to those in wild-type mice (Table 2). NAD and FADare central biomolecules involved in energy production and mitochondrialmetabolic activity; declines in NAD and FAD levels reflect decreasedmitochondrial number, density, and activity. Thus, the findings here inparallel demonstrated that mtHtt-induced mitochondria-accumulated VCPcauses mitochondrial dysfunction and global energy deficits in HD,leading to neuronal cell death. Because the depletion in NAD was notedin HD patient cells and blood, normalization of NAD content in HD mouseplasma by HV-3 treatment might provide a biomarker amenable fortherapeutic intervention.

TABLE 1 Content of Mitochondiral intermediates in HD R6/2 mouse plasmaConcentration (μg/ml mouse plasma) -Acetyl- Group Asparate AspartalCis-Aconitate Citrate Ketoglutarate Malate Wt/Veh 1.31 ± 0.31 0.93 ±0.08 0.49 ± 0.05 31.31 ± 2.18 11.25 ± 1.70  3.36 ± 0.41 Wt/HV-3 0.98 ±0.20 0.98 ± 0.14 0.45 ± 0.05 33.66 ± 2.74 11.66 ± 2.15  4.45 ± 0.64HD/Veh 1.31 ± 0.19 0.75 ± 0.08 0.47 ± 0.06 24.52 ± 1.48 6.23 ± 1.11 3.31± 0.43 HD/HV3 1.90 ± 0.23 0.84 ± 0.07 0.48 ± 0.05 31.03 ± 2.02# 5.88 ±0.63 4.07 ± 0.46 Concentration (μg/ml mouse plasma) Group SuccinateFumarate Pyruvate Isocitrate NAD+ FAD Wt/Veh 15.63 ± 1.79  3.14 ± 0.347.05 ± 0.78 5.66 ± 0.56 0.07 ± 0.01 0.25 ± 0.02 Wt/HV-3 18.87 ± 2.12 3.84 ± 0.51 10.30 ± 1.15  7.24 ± 0.78 0.07 ± 0.01 0.29 ± 0.02 HD/Veh10.32 ± 1.34* 2.57 ± 0.34 12.71 ± 1.32* 8.12 ± 0.80* 0.04 ± 0.004* 0.20± 0.02* HD/HV3 9.07 ± 1.36 3.32 ± 0.41 13.74 ± 3.19  9.63 ± 1.05 0.07 ±0.01# 0.28 ± 0.03# *p < 0.05 vs. wildtype mice treated with TAT; #p <0.0 vs. HD mice treated with TAT

Table 1 listed the changes of intermediates of mitochondrial TCA cyclein HD R6/2 mouse plasma. Sixteen intermediates derived frommitochondrial TCA cycle were determined by LC/MS/MS using the standardsubstrates. We found that 8 intermediates derived from the TCA cyclewere decreased in HD R6/2 mouse plasma relative to those from wild-typelittermates. HV-3 treatment specifically corrected the amount of NAD⁺,FAD, and citrate. In addition, four intermediates (acetyl-CoA,succinyl-CoA, oxaloacetate, and ketobutyrate) were not detectable in themouse plasma. Data are mean±SE, n=15 mice/group. Data are analyzed byone-way ANOVA with post hoc Turkey test.

TABLE 2 Concentration of Intermediates of amino acid metabolism in HDR6/2 mouse plasma Concentration in mouse plasma (μM) Alanine SerineThreonine Proline Valine WT 153.6 +/− 8.5  61.5 +/− 5.3 36.6 +/− 3.188.2 +/− 8.2 50.6 +/− 5.6 Wt/HV-3 130.0 +/− 14.7 56.1 +/− 5.8 31.3 +/−2.6  90.3 +/− 16.3 39.5 +/− 2.8 HD 149.6 +/− 19.4  120.9 +/− 23.6*  54.3+/− 9.3*  167.4 +/− 22.6* 62.2 +/− 7.9 HD/HV3 169.0 +/− 16.0 127.4 +/−9.5  67.3 +/− 7.8 171.8 +/− 7.2  80.0 +/− 8.1 WT 250.8 +/− 18.0 11.7 +/−2.1 13.7 +/− 0.9 77.5 +/− 5.8 31.9 +/− 1.9 Wt/HV-3 242.9 +/− 17.2  7.8+/− 1.9 14.2 +/− 1.1 78.1 +/− 5.7 33.6 +/− 1.9 HD  354.2 +/− 26.9*  8.1+/− 4.8  17.0 +/− 1.1* 103.7 +/− 5.3*  39.9 +/− 1.9* HD/HV3 376.6 +/−17.5 10.6 +/− 2.7 19.9 +/− 1.3 102.8 +/− 3.2  43.8 +/− 1.4 Concentrationin mouse plasma (μM) Iso- Aspartic Leucine Leucine Acid Glutamine WT11.3 +/− 1.4 64.2 +/− 5.9 3.0 +/− 0.6 394.1 +/− 30.6 Wt/HV-3 13.8 +/−1.6 72.2 +/− 5.9 3.2 +/− 0.4 370.0 +/− 15.3 HD  17.5 +/− 2.3*  93.9 +/−10.9* 4.6 +/− 0.5  637.7 +/− 77.1* HD/HV3 15.5 +/− 1.7 84.3 +/− 6.1 4.1+/− 0.3 627.7 +/− 45.1 WT 158.6 +/− 19.1 35.6 +/− 2.8 19.9 +/− 2.5  28.4+/− 4.2 Wt/HV-3 162.8 +/− 19.3 35.6 +/− 2.6 19.8 +/− 2.0  29.2 +/− 2.9HD  365.2 +/− 48.1* 43.4 +/− 2.5 25.3 +/− 2.9   28.2 +/− 3.5* HD/HV3345.5 +/− 18.4 42.5 +/− 2.1 31.4 +/− 2.9  25.2 +/− 2.2 *p < 0.05 vs.wildtype mice treated with TAT

Table 2 listed the intermediates of amino acid metabolic pathway.Eighteen intermediates derived from amino acid metabolism weredetermined by LC/MS/MS method. HV-3 treatment had no statistic effectson these intermediates. These data further demonstrated a selective roleof HV-3 on mitochondrial energy metabolic activity. Data are mean±SE,n=10 mice/group. Data are analyzed by one-way ANOVA with post hoc Turkeytest.

HD R6/2 mice and wild-type littermates were treated with control peptideTAT or HV-3 peptides at 3 mg/kg/day from the age of 5 weeks to 13 weeks.Plasma was harvested at the age of 12 weeks. Target metabolomicsanalysis of mouse plasma was conducted as described in Method. In thisstudy, we focused on mitochondrial TCA cycle and amino acid metabolicpathways for the analysis, because failures in these two pathways in HDpatients were previously reported.

Method

Target Metabolomics Analysis

Mouse plasma was harvested at the age of 12 weeks from HD R6/2 mice andwild-type littermates. Mouse plasma was mixed with internal standardsfollowed by an addition of methanol to precipitate the sample protein bycentrifuging at 12,000 rpm. After centrifugation, supernatant was driedunder nitrogen flow and the dried pellet was suspended using 80 μl HPLCgrade water. Twenty μl of suspended sample was injected into the HPLCsystem which is directly interfaced with a triple-quadrupole massspectrometer. The HPLC eluent was automatically injected into the massspectrometer and the compounds in the eluent were analyzed withelectrospray ionization (at positive or negative charged) tandem massspectrometry using the scan termed multiple reaction monitoring. Theanalysis was conducted at the Cleveland Clinic Lerner Research CenterMetabolomics core facility by individuals blind to the treatment groups.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All references,publications, and patents cited in the present application are hereinincorporated by reference in their entirety.

Having described the invention, I claim:
 1. A composition comprising: a therapeutic peptide consisting of the amino acid sequence that is at least about 75% identical to SEQ ID NO: 4; and a cell-penetrating peptide transport moiety that is linked to the therapeutic peptide.
 2. The method of claim 1, wherein the therapeutic peptide has the amino acid sequence of SEQ ID NO:
 3. 3. The composition of claim 1, wherein the cell-penetrating peptide transport moiety is an HIV Tat transport moiety.
 4. A composition comprising: a therapeutic peptide consisting of the amino acid sequence at least 75% identical to SEQ ID NO: 4 that inhibits the binding or complexing of VCP with mutant huntingtin protein (mtHtt); and a cell-penetrating peptide transport moiety that is linked to the therapeutic peptide.
 5. The composition of claim 4, wherein the therapeutic peptide has the amino acid sequence of SEQ ID NO:
 3. 6. The composition of claim 4, wherein the cell-penetrating peptide transport moiety is an HIV-TAT transport moiety.
 7. A composition comprising: a therapeutic peptide consisting of the amino acid sequence that is at least about 75% identical to SEQ ID NO: 4; and an HIV-TAT transport moiety.
 8. The composition of claim 7, wherein the therapeutic peptide has the amino acid sequence of SEQ ID NO:
 3. 